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Myocardin is a potent transcriptional coactivator protein, which functions as the master regulator of vascular smooth muscle cell differentiation. The cofactor activity of myocardin is mediated by its physical interaction with serum response factor, a ubiquitously expressed transactivator that binds to CArG boxes in genes encoding smooth muscle-restricted proteins. Purine-rich element binding protein B (Purβ) represses the transcription of the smooth muscle α-actin gene (Acta2) in fibroblasts and smooth muscle cells by interacting with single-stranded DNA sequences flanking two 5' CArG boxes in the Acta2 promoter. In this study, the ability of Purβ to modulate the cofactor activity of myocardin was investigated using a combination of cellular and biochemical approaches. Results of smooth muscle gene promoter-reporter assays indicated that Purβ specifically inhibits the coactivator function of myocardin in a manner requiring the presence of all three single-stranded DNA binding domains in the Purβ homodimer. DNA binding analyses demonstrated that Purβ interacts with CArG-containing DNA elements with a much lower affinity compared to other purine-rich target sequences present in the Acta2 promoter. Co-immunoprecipitation and DNA pull-down assays revealed that Purβ associates with myocardin and serum response factor when free or bound to duplex DNA containing one or more CArG boxes. Functional analysis of engineered Purβ point mutants identified several amino acid residues essential for suppression of myocardin activity. Collectively, these findings suggest an inhibitory mechanism involving direct protein-protein interaction between the homodimeric Purβ repressor and the myocardin-serum response factor-CArG complex.

Myocardin is a potent transcriptional coactivator protein, which functions as the master regulator of vascular smooth muscle cell differentiation. The cofactor activity of myocardin is mediated by its physical interaction with serum response factor, a ubiquitously expressed transactivator that binds to CArG boxes in genes encoding smooth muscle-restricted proteins. Purine-rich element binding protein B (Purβ) represses the transcription of the smooth muscle α-actin gene (Acta2) in fibroblasts and smooth muscle cells by interacting with single-stranded DNA sequences flanking two 5' CArG boxes in the Acta2 promoter. In this study, the ability of Purβ to modulate the cofactor activity of myocardin was investigated using a combination of cellular and biochemical approaches. Results of smooth muscle gene promoter-reporter assays indicated that Purβ specifically inhibits the coactivator function of myocardin in a manner requiring the presence of all three single-stranded DNA binding domains in the Purβ homodimer. DNA binding analyses demonstrated that Purβ interacts with CArG-containing DNA elements with a much lower affinity compared to other purine-rich target sequences present in the Acta2 promoter. Co-immunoprecipitation and DNA pull-down assays revealed that Purβ associates with myocardin and serum response factor when free or bound to duplex DNA containing one or more CArG boxes. Functional analysis of engineered Purβ point mutants identified several amino acid residues essential for suppression of myocardin activity. Collectively, these findings suggest an inhibitory mechanism involving direct protein-protein interaction between the homodimeric Purβ repressor and the myocardin-serum response factor-CArG complex.

Purine-rich element-binding protein B (Purβ) inhibits myofibroblast differentiation by repressing the expression of the smooth muscle α-actin gene (Acta2). Several reports have identified the structural domains in Purβ that enable its characteristic interaction with purine-rich single-stranded DNA (ssDNA) sequences in the Acta2 promoter. However, little is known about the physical and functional effects of single nucleotide polymorphisms that alter individual amino acid residues in Purβ. This study evaluated seven rare single amino acid variants of human PURB engineered into the homologous mouse Purβ protein. Mapping the location of variant residues on a homology model of the Purβ homodimer suggested that most of the altered residues are remote from the predicted ssDNA-binding regions of the protein. The repressor activity of each Purβ variant was assessed in transfected fibroblasts and smooth muscle cells via Acta2 promoter-reporter assays. A Q64* nonsense variant was completely inactive while missense variants exhibited repressor activity that ranged from ~1.5-fold greater to ~2-fold less than wild-type Purβ. Lower activity variants P223L and R297Q were expressed in bacteria and purified to homogeneity. Each variant was physically indistinguishable from wild-type Purβ in terms of quaternary structure and thermostability. Results of DNA and protein binding assays indicated that the P223L and R297Q variants retained high affinity and specificity for purine-rich ssDNA sequences but differed in their interaction with other Acta2 regulatory proteins. These findings suggest that the presence of certain variant residues affects the Acta2 repressor activity of Purβ by altering its interaction with other transcription factors but not with ssDNA.

Acute myelogenous leukemia (AML) is an aggressive hematologic cancer characterized by infiltration of proliferative, clonal, abnormally differentiated cells of myeloid lineage in the bone marrow and blood. Malignant cells in AML often exhibit chromosomal and other genetic or epigenetic abnormalities that are useful in prognostic risk assessment. In this study, the relative expression and novel single-stranded DNA (ssDNA) binding function of purine-rich element binding proteins A and B (Purα and Purβ) were systematically evaluated in established leukemia cell lines and in lineage committed myeloid cells isolated from patients diagnosed with a hematologic malignancy. Western blotting revealed that Purα and Purβ are markedly elevated in CD33+ /CD66b+ cells from AML patients compared to healthy subjects and to patients with other types of myeloid cell disorders. Results of in silico database analysis of PURA and PURB mRNA expression during hematopoiesis in conjunction with the quantitative immunoassay of the ssDNA-binding activities of Purα and Purβ in transformed leukocyte cell lines pointed to Purβ as the more distinguishing biomarker of myeloid cell differentiation status. Purβ ssDNA-binding activity was significantly increased in myeloid cells from AML patients but not from individuals with other myeloid-related diseases. The highest levels of Purβ activity were detected in myeloid cells from primary AML patients and from AML patients displaying other risk factors forecasting a poor prognosis. Collectively, these findings suggest that the enhanced ssDNA-binding activity of Purβ in transformed myeloid cells may serve as a unique and measurable phenotypic trait for improving prognostic risk stratification in AML.

Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+SAntilles, and SS‐BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr‐1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL‐1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro‐inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM‐1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent—and possibly dominant—role for an abnormal monocyte‐TNF‐endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal.

Myofibroblast differentiation is characterized by an increased level of expression of cytoskeletal smooth muscle α-actin. In human and murine fibroblasts, the gene encoding smooth muscle α-actin (Acta2) is tightly regulated by a network of transcription factors that either activate or repress the 5' promoter-enhancer in response to environmental cues signaling tissue repair and remodeling. Purine-rich element-binding protein B (Purβ) suppresses the expression of Acta2 by cooperatively interacting with the sense strand of a 5' polypurine sequence containing an inverted MCAT cis element required for gene activation. In this study, we evaluated the chemical basis of nucleoprotein complex formation between the Purβ repressor and the purine-rich strand of the MCAT element in the mouse Acta2 promoter. Quantitative single-stranded DNA (ssDNA) binding assays conducted in the presence of increasing concentrations of monovalent salt or anionic detergent suggested that the assembly of a high-affinity nucleoprotein complex is driven by a combination of electrostatic and hydrophobic interactions. Consistent with the results of pH titration analysis, site-directed mutagenesis revealed several basic amino acid residues in the intermolecular (R267) and intramolecular (K82 and R159) subdomains that are essential for Purβ transcriptional repressor function in Acta2 promoter-reporter assays. In keeping with their diminished Acta2 repressor activity in fibroblasts, purified Purβ variants containing an R267A mutation exhibited reduced binding affinity for purine-rich ssDNA. Moreover, certain double and triple-point mutants were also defective in binding to the Acta2 corepressor protein, Y-box-binding protein 1. Collectively, these findings establish the repertoire of noncovalent interactions that account for the unique structural and functional properties of Purβ.

A hallmark of dysfunctional fibroblast to myofibroblast differentiation associated with fibrotic disorders is persistent expression of ACTA2, the gene encoding the cyto-contractile protein smooth muscle α-actin. In this study, a PURB-specific gene knockdown approach was used in conjunction with biochemical analyses of protein subdomain structure and function to reveal the mechanism by which purine-rich element binding protein B (Purβ) restricts ACTA2 expression in mouse embryo fibroblasts (MEFs). Consistent with the hypothesized role of Purβ as a suppressor of myofibroblast differentiation, stable short hairpin RNA-mediated knockdown of Purβ in cultured MEFs promoted changes in cell morphology, actin isoform expression, and cell migration indicative of conversion to a myofibroblast-like phenotype. Promoter-reporter assays in transfected Purβ knockdown MEFs confirmed that these changes were attributable, in part, to de-repression of ACTA2 transcription. To map the domains in Purβ responsible for ACTA2 repression, several recombinant truncation mutants were generated and analyzed based on hypothetical, computationally-derived models of the tertiary and quaternary structure of Purβ. Discrete subdomains mediating sequence- and strand-specific cis-element binding, protein-protein interaction, and inhibition of a composite ACTA2 enhancer were identified using a combination of biochemical, biophysical, and cell-based assays. Our results indicate that the Purβ homodimer possesses three separate but unequal single-stranded DNA-binding modules formed by subdomain-specific inter- and intramolecular interactions. This structural arrangement suggests that the cooperative assembly of the dimeric Purβ repressor on the sense strand of the ACTA2 enhancer is dictated by the association of each subdomain with distinct purine-rich binding sites within the enhancer.

Peri-transplant surgical trauma and ischemia/reperfusion injury in accepted murine heterotopic heart grafts has been associated with myofibroblast differentiation, cardiac fibrosis and biomechanical-stress activation of the fetal myocardial smooth muscle α-actin (SMαA) gene. The wound-healing agonists, transforming growth factor β1 and thrombin, are known to coordinate SMαA mRNA transcription and translation in activated myofibroblasts by altering the subcellular localization and mRNA-binding affinity of the Y-box binding protein-1 (YB-1) cold-shock domain (CSD) protein that governs a variety of cellular responses to metabolic stress. YB-1 accumulated in polyribosome-enriched regions of the sarcoplasm proximal to cardiac intercalated discs in accepted heart grafts. YB-1 binding to a purine-rich motif in exon 3 of SMαA mRNA that regulates translational efficiency increased substantially in perfusion-isolated, rod-shaped adult rat cardiomyocytes during phenotypic de-differentiation in the presence of serum-derived growth factors. Cardiomyocyte de-differentiation was accompanied by the loss of a 60 kDa YB-1 variant that was highly expressed in both adult myocardium and freshly isolated myocytes and replacement with the 50 kDa form of YB-1 (p50) typically expressed in myofibroblasts that demonstrated sequence-specific interaction with SMαA mRNA. Accumulation of p50 YB-1 in reprogrammed, de-differentiated myocytes was associated with a 10-fold increase in SMαA protein expression. Endomyocardial biopsies collected from patients up to 14 years after heart transplant showed variable yet coordinately elevated expression of SMαA and p50 YB-1 protein and demonstrable p50 YB-1:SMαA mRNA interaction. The p60 YB-1 variant in human heart graft samples, but neither mouse p60 nor mouse or human p50, reacted with an antibody specific for the phosphoserine 102 modification in the YB-1 CSD. Modulation of YB-1 subcellular compartmentalization and mRNA-binding activity may be linked with reprogramming of contractile protein gene expression in ventricular cardiomyocytes that could contribute to maladaptive remodeling in accepted, long-term heart grafts.

We previously observed gross hemorrhage in plasminogen activator inhibitor type-1 (PAI-1) knockout (PKO) mice with induced myocardial infarction (MI). We hypothesized that it reflected degradation of vessels - a phenomenon we termed vascular rhexis. Accordingly, in the present study we characterized vascular rhexis in C57BL6 mice. MI was induced in 10- to 12-week-old mice by coronary artery ligation for 24, 48, 72 or 96 h. Hemorrhage was quantified by non-cross-reacting enzyme-linked immunosorbent assay of left ventricular (LV) hemoglobin corrected for myoglobin. Degradation of vasculature was quantified by the appearance of alpha smooth muscle actin (alphaSMA) in low salt soluble fractions of LV homogenates (Western blotting) and by immunohistochemistry (residual alphaSMA). Co-staining for CD31 (endothelial cells) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) (a marker of cell death) was used to identify capillary rhexis. PKO mice (n = 9) had marked hemorrhage in infarct zones (432 +/- 27 standard error of mean microL blood/g). Hemorrhage was evident in C57BL6 mice as well (n = 6): 51 +/- 8 microL/g LV 96 h after coronary occlusion compared with 10 +/- 5 microL /g, n = 13 in normal LVs. Residual intact vasculature was reduced 48 h after infarction. Thus, an average of 16 +/- 1.6 small- and medium-sized vessels (n = 5 hearts) were seen compared with 84 +/- 4.8 in normal LVs (n = 3, Por = 0.05). An approximately three-fold increase in soluble alphaSMA 48 h after MI (2.68 +/- 0.28, n = 6) was seen relative to that in normal LVs defined as 1.0 +/- 0.04, n = 10, Por = 0.05. Capillary degradation was evident as well, as judged from CD31 and TUNEL co-localization. Vascular rhexis occurs within 48 h after the onset of MI. It may contribute to the early no-reflow phenomenon and to late negative LV remodeling.

Sickle cell anemia is accompanied by the activation of coagulation and thrombosis. We have studied the abnormal expression of tissue factor (TF) by the pulmonary vein endothelium of the mild-phenotype NY1DD sickle transgenic. As detected by immunofluorescence microscopy, this occurs only after the NY1DD mouse is exposed to hypoxia/reoxygenation (H/R), which actually causes ischemia/reperfusion in the sickle cell disease—but not the normal—mouse model. We tested the hypothesis that the nuclear factor-kappa B (NFκB)-activating inflammation that develops in post-H/R NY1DD mice is responsible for this phenotype switch. Various NFκB inhibitors (including p50-specific andrographolide) demonstrated that endothelial TF positivity is NFκB dependent. Several systemic inflammatory stimulators (tumor necrosis factor [TNFα], lipopolysaccharide, thioglycollate, and carageenan) given to control mice showed that the inflammatory promotion of TF expression by only pulmonary vein endothelium is not specific to the sickle cell disease model. We bred the NFκB(p50)-/- state into the NY1DD mouse. Combined with marrow transplantation, this allowed the creation of NY1DD mice that were NFκB(p50)-/- only in peripheral blood cells (and marrow) versus only in vessel walls (and tissues). This process revealed that endothelial TF expression in the NY1DD mouse is highly dependent on NFκB(p50) in peripheral blood mononuclear cells—but not in the vessel wall. In confirmation, the infusion of post-H/R sickle mouse blood mononuclear cells into naive NY1DD mice stimulated endothelial TF expression; the infusion of such cells from unstimulated sickle cell disease mice at ambient air did not stimulate TF expression. We conclude that peripheral blood mononuclear cells indirectly promote endothelial TF expression via a NFκB(p50)-dependent mechanism. This approach may be relevant to the role of coagulopathy in clinical sickle cell disease.

The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.

Gonadotropin-releasing hormone-1 (GnRH1) controls reproduction by stimulating the release of gonadotropins from the pituitary. To characterize regulatory factors governing GnRH1 gene expression, we employed biochemical and bioinformatics techniques to identify novel GnRH1 promoter-binding proteins from the brain of the cichlid fish, Astatotilapia burtoni (A. burtoni). Using an in vitro DNA-binding assay followed by mass spectrometric peptide mapping, we identified two members of the purine-rich element-binding (Pur) protein family, Puralpha and Purbeta, as candidates for GnRH1 promoter binding and regulation. We found that transcripts for both Puralpha and Purbeta colocalize in GnRH1-expressing neurons in the preoptic area of the hypothalamus in A. burtoni brain. Furthermore, we confirmed in vivo binding of endogenous Puralpha and Purbeta to the upstream region of the GnRH1 gene in A. burtoni brain and mouse neuronal GT1-7 cells. Consistent with the relative promoter occupancy exhibited by endogenous Pur proteins, overexpression of Purbeta, but not Puralpha, significantly downregulated GnRH1 mRNA levels in transiently transfected GT1-7 cells, suggesting that Purbeta acts as a repressor of GnRH1 gene transcription.

Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related microRNAs (miRNAs) within their introns, which, in turn, control muscle myosin content, myofiber identity and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, co-expresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 are functionally redundant, and play a dominant role in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance.

Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins, Purα and Purβ, in transcriptional repression of the smooth muscle α-actin gene in vascular cell types. Although Purα and Purβ share substantial sequence homology and nucleic acid binding properties, genomic promoter and cis-element occupancy studies reported herein suggest that Purβ is the dominant factor in gene regulation. To dissect the molecular basis of Purβ repressor activity, site-directed mutagenesis was used to map amino acids critical to the physical and functional interaction of Purβ with the smooth muscle α-actin promoter. Of all the various acidic, basic, and aromatic residues studied, mutation of positionally conserved arginines in the class I or class II repeat modules significantly attenuated Purβ repressor activity in transfected vascular smooth muscle cells and fibroblasts. DNA binding and protein-protein interaction assays were conducted with purified recombinant Purβ and selected mutants to reveal the physical basis for loss-of-function. Mutants R57E, R57E/R96E, and R57A/R96A each exhibited reduced single-stranded DNA binding affinity for an essential promoter element and diminished interaction with corepressor YB-1/MSY1. Structural analyses of the R57A/R96A and R57E/R96E double mutants in comparison to the wild-type Purβ homodimer revealed aberrant self-association into higher order oligomeric complexes, which correlated with decreased α-helical content and defective DNA and protein binding in vitro. These findings point to a previously unrecognized structural role for certain core arginine residues in forming a conformationally stable Purβ protein capable of physical interactions necessary for smooth muscle α-actin gene repression.

Adult skeletal muscle retains the capability of transcriptional reprogramming. This attribute is readily observable in the non-weight-bearing (NWB) soleus muscle, which undergoes a slow-to-fast fiber type transition concurrent with decreased beta-myosin heavy chain (betaMyHC) gene expression. Our previous work showed that Sp3 contributes to decreased betaMyHC gene expression under NWB conditions. In this study, we demonstrate that physical and functional interactions between Sp3, Puralpha, and Purbeta proteins mediate repression of betaMyHC expression under NWB conditions. Binding of Puralpha or Purbeta to the single-stranded betaMyHC distal negative regulatory element-sense strand (dbetaNRE-S) element is markedly increased under NWB conditions. Ectopic expression of Puralpha and Purbeta decreased betaMyHC reporter gene expression, while mutation of the dbetaNRE-S element increased expression in C2C12 myotubes. The dbetaNRE-S element conferred Pur-dependent decreased expression on a minimal thymidine kinase promoter. Short interfering RNA sequences specific for Sp3 or for Puralpha and Purbeta decreased endogenous Sp3 and Pur protein levels and increased betaMyHC reporter gene expression in C2C12 myotubes. Immunoprecipitation assays revealed an association between endogenous Puralpha, Purbeta, and Sp3, while chromatin immunoprecipitation assays demonstrated Puralpha, Purbeta, and Sp3 binding to the betaMyHC proximal promoter region harboring the dbetaNRE-S and C-rich elements in vivo. These data demonstrate that Pur proteins collaborate with Sp3 to regulate a transcriptional program that enables muscle cells to remodel their phenotype.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31) is known to inhibit platelet function and thrombus formation. The mechanisms involved in PECAM-1's roles as a modulator of hemostasis are still not completely understood. We examined the role of PECAM-1 as a regulator of tissue factor (TF) expression, a known important inducer of thrombosis. Wildtype and CD31KO mice underwent transient (30 min) renal ischemia followed by 24 h re-perfusion and their kidneys assessed for apoptosis, fibrin formation, and tissue factor expression. CD31KO mice exhibited increased tubular epithelial and endothelial apoptosis, increased fibrin deposition, and tissue factor expression. Human umbilical vein endothelial cells (HUVEC) transfected with antisense (AS) PECAM-1 oligonucleotides to downregulate PECAM-1 expression, exhibited greater induction of TF mRNA and protein expression as well as increased expression and nuclear localization of the transcription factor Egr-1 compared to scrambled AS PECAM-1 (Scr)-treated HUVEC following thrombin stimulation. TF induction was found to be mediated through thrombin receptor PAR-1 and the Galphai/o subunit of G-protein, confirmed by PAR-1 antagonist and pertussis toxin inhibition respectively. Thrombin-mediated TF induction was dependent on Rho Kinase activity, phosphorylation of p38(MAPK) and p85 & Akt dephosphorylation. The inverse correlation of PI3K-Akt phosphorylation with p38 (MAPK) phosphorylation was confirmed by pharmacological inhibition. These studies suggest that PECAM-1 is involved in regulating a signaling pathway, affecting PI3K and Akt activation, p38 (MAPK) phosphorylation, which in turn, affects Egr-1 expression and nuclear translocation, ultimately affecting TF expression. These findings provide new insights into the action of PECAM-1 as a modulator of thrombosis.

Purbeta is a gene regulatory factor belonging to a family of highly conserved nucleic acid-binding proteins related by their ability to preferentially bind single-stranded DNA or RNA sequences rich in purine nucleotides. In conjunction with Puralpha, Purbeta has been implicated in transcriptional and translational repression of genes encoding contractile proteins found in the heart and vasculature. Although several models of sequence-specific DNA recognition, strand separation, and activator inhibition by oligomeric Puralpha and Purbeta have been proposed, it is currently unclear whether protein-protein interaction is a prerequisite to, or a consequence of nucleic acid binding. In this study, a recombinant protein purification scheme was devised to yield homogenous mouse Purbeta devoid of nucleic acid. Recombinant Purbeta was then subjected to light scattering and analytical ultracentrifugation analyses to assess the size, shape, and oligomeric state of the purified protein in solution. Results of laser light scattering and sedimentation velocity experiments indicated that Purbeta reversibly self-associates in the absence of nucleic acid. Both approaches independently showed that the hydrodynamic shape of the Purbeta homodimer is markedly asymmetric and non-spherical. Sedimentation velocity analyses indicated that dimeric Purbeta has a sedimentation coefficient of 3.96 Svedberg, a frictional coefficient ratio (f/f(0)) of 1.60, and a hydrodynamic radius of 4.43 nm. These values were consistent with those determined by independent dynamic light scattering studies. Sedimentation equilibrium analyses confirmed that Purbeta self-associates in a reversible monomer-dimer equilibrium characterized by a K(d) = 1.13 +/- 0.27 microm.

Objective— Proliferation of vascular smooth muscle cells (VSMCs) contributes to restenosis after coronary intervention. We have shown previously that increased expression of plasminogen activator inhibitor type 1 (PAI-1) limits VSMC apoptosis. Because apoptosis and proliferation appear to be linked, we sought to determine whether increased PAI-1 would affect VSMC proliferation. Methods and Results— VSMCs were explanted from control and transgenic mice (SM22-PAI + ) in which VSMC expression of PAI-1 was increased. Increased growth of SM22-PAI + -VSMCs (2.3±0.4-fold) reflected, at least partially, increased proliferation. Greater expression of FLICE-like inhibitory protein (FLIP; 2.7-fold) and its cleaved active form were seen in SM22-PAI + -VSMCs. The balance between caspase-8 and FLIP favored proliferation in SM22-PAI + -VSMCs. Increased expression of NF-κB and activation of extracellular signal-regulated kinase (ERK) were demonstrated in SM22-PAI + -VSMCs (fold=NF-κB=2.2±0.1, fold=phosphorylated-ERK=1.6±0.1). Results were confirmed when expression of PAI-1 was increased by transfection. Inhibition of NF-κB and ERK attenuated proliferation in SM22-PAI + -VSMCs. Increased expression of PAI-1 promoted proliferation when VSMCs were exposed to tumor necrosis factor (TNF). Conclusions— Increased expression of PAI-1 is associated with greater activity of FLIP that promotes VSMC proliferation through NF-κB and ERK. Thus, when vascular wall expression of PAI-1 is increased, restenosis after coronary intervention is likely to be potentiated by greater proliferation of VSMC and resistance to apoptosis.

Profibrotic regulatory mechanisms for tissue repair after traumatic injury have developed under strong evolutionary pressure to rapidly stanch blood loss and close open wounds. We have examined the roles played by two profibrotic mediators, transforming growth factor beta1 (TGFbeta1) and thrombin, in directing expression of the vascular smooth muscle alpha-actin (SMalphaA) gene, an important determinant of myofibroblast differentiation and early protein marker for stromal cell response to tissue injury. TGFbeta1 is a well known transcriptional activator of the SMalphaA gene in myofibroblasts. In contrast, thrombin independently elevates SMalphaA expression in human pulmonary myofibroblasts at the posttranscriptional level. A common feature of SMalphaA up-regulation mediated by thrombin and TGFbeta1 is the involvement of the cold shock domain protein YB-1, a potent repressor of SMalphaA gene transcription in human fibroblasts that also binds mRNA and regulates translational efficiency. YB-1 dissociates from SMalphaA enhancer DNA in the presence of TGFbeta1 or its Smad 2, 3, and 4 coregulatory mediators. Thrombin does not effect SMalphaA gene transcription but rather displaces YB-1 from SMalphaA exon 3 coding sequences previously shown to be required for mRNA translational silencing. The release of YB-1 from promoter DNA coupled with its ability to bind RNA and shuttle between the nucleus and cytoplasm is suggestive of a regulatory loop for coordinating SMalphaA gene output in human pulmonary myofibroblasts at both the transcriptional and translational levels. This loop may help restrict organ-destructive remodeling due to excessive myofibroblast differentiation.

Plasticity of smooth muscle alpha-actin gene expression in fibroblasts and vascular smooth muscle cells is mediated by opposing effects of transcriptional activators and repressors. Among these factors, three single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1, have been implicated as coregulators of a cryptic 5'-enhancer module. In this study, a molecular analysis of Purbeta, the least well characterized member of this group, was conducted. Southwestern and Northwestern blotting of purified Purbeta deletion mutants using smooth muscle alpha-actin-derived probes mapped the minimal single-stranded DNA/RNA-binding domain to a conserved region spanning amino acids 37-263. Quantitative binding assays indicated that the relative affinity and specificity of Purbeta for single-stranded DNA were influenced by purine/pyrimidine content; by non-conserved regions outside amino acids 37-263; and by cell-derived proteins, specifically MSY1. When overexpressed in A7r5 vascular smooth muscle cells, Purbeta (but not Puralpha) inhibited transcription of a smooth muscle-specific mouse alpha-actin promoter transgene. Structural domains required for Purbeta repressor activity included the minimal DNA-binding region and a C-terminal domain required for stabilizing high affinity protein and nucleic acid interactions. Purbeta inhibitory activity in transfected A7r5 cells was potentiated by MSY1, but antagonized by serum response factor, reinforcing the idea that interplay among activators and repressors may account for phenotypic changes in smooth muscle alpha-actin-expressing cell types.

Objective: Chronic rejection in cardiac allografts depletes vascular smooth muscle (VSM) α-actin from the coronary arterial smooth muscle bed while promoting its abnormal accumulation in cardiomyocytes and myofibroblasts. The objective was to determine if the newly discovered TEF1, MSY1, Purα and Purβ VSM α-actin transcriptional reprogramming proteins (TRPs) were associated with development of chronic rejection histopathology in accepted murine cardiac allografts. Methods: A mouse heterotopic cardiac transplant model was employed using H2 locus-mismatched mouse strains (DBA/2 or FVB/N to C57BL/6). Recipients were immunosuppressed to promote long-term allograft acceptance and emergence of chronic rejection. Explanted grafts and isolated heart cells were evaluated for changes in the DNA-binding activity and subcellular distribution of VSM α-actin transcriptional regulatory proteins. Results: The DNA-binding activity of all four TRPs was high in the developing mouse ventricle, minimal in adult donor hearts and increased substantially within 30 days after transplantation. Immunohistologic analysis revealed nuclear localization of Purβ and MSY1 particularly in fibrotic areas of the allograft myocardium demonstrating extravascular accumulation of VSM α-actin. Cardiomyocytes isolated from adult, non-transplanted mouse hearts not only exhibited less VSM α-actin expression and lower levels of TRPs compared to isolated cardiac fibroblasts or neonatal cardiomyocytes, but also contained a novel size variant of the MSY1 protein. Conclusion: Accumulation of TRPs in cardiac allografts, particularly within the fibroblast-enriched myocardial interstitium, was consistent with their potential role in VSM α-actin gene reprogramming, fibrosis and dysfunctional remodeling following transplant. These nuclear protein markers could help stage peri-transplant cellular events that precede formation of graft-destructive fibrosis and coronary vasculopathy during chronic rejection.

An asymmetric polypurine-polypyrimidinecis-element located in the 5′ region of the mouse vascular smooth muscle α-actin gene serves as a binding site for multiple proteins with specific affinity for either single- or double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to trans-activate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Purα and Purβ with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Purα, Purβ, and MSY1in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Purα, Purβ, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Purα, Purβ, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.

Tissue factor, the cellular initiator of blood coagulation, has been implicated as a determinant of metastatic potential in human melanoma cells. Here, we report that differential expression of tissue factor in murine melanoma cell lines of known metastatic behavior is mediated by AP-1-dependent and 12S E1A oncoprotein-repressible gene transcription. When compared to weakly metastatic C10 cells, highly metastatic M4 cells possessed elevated levels of tissue factor cofactor activity, transfected promoter activity, and heterodimeric AP-1 DNA-binding complexes containing Fra-1. Transient co-expression of the adenovirus E1A 12S oncoprotein strongly repressed transcription of an AP-1-driven tissue factor reporter gene indicating the additional requirement of N-terminal E1A-interacting coactivators. Stable expression of E1A mutants defective in CBP/p300-binding failed to suppress tissue factor expression and experimental metastasis by M4 cells while clones expressing wild type E1A exhibited greatly reduced tissue factor cofactor activity and metastatic potential in vivo. Overexpression of functional tissue factor in cells containing wild type E1A failed to restore the highly metastatic M4 phenotype suggesting that additional E1A-responsive and CBP/p300-dependent genes are required to facilitate metastasis of murine melanoma cells demonstrating high tissue factor expression and cofactor activity. J. Cell. Biochem. 85: 54–71, 2002. © 2002 Wiley-Liss, Inc.

Expression of smooth muscle alpha-actin (SMαA) is essential for myofibroblast-mediated wound contraction following tissue injury. The Pur α/β and YB-1 transcriptional repressors govern the DNA-binding activity of serum response factor (SRF) and phosphorylated Smad3 (pSmad3) transcriptional activators during induction of SMαA gene expression in human pulmonary myofibroblasts. In quiescent fibroblasts, Pur α exhibited a novel function in enhancing stability of pre-existing SRF complexes with SMαA core promoter DNA, whereas Pur β was more effective in disrupting SRF-DNA interaction. Pur proteins were less efficient competitors of pre-existing, core-promoter complexes containing both SRF and pSmad3 in nuclear extracts from TGFβ1-activated myofibroblasts. TGFβ1 signaling dissociated a SRF/Pur protein complex with concurrent formation of a transient pSmad3/MRTF-A/Pur β complex during early phase myofibroblast differentiation. Pur β was replaced by Pur α in the pSmad3/MRTF-A complex in mature myofibroblasts. Combining all three repressors potently inhibited SRF and pSmad3 binding to promoter DNA in quiescent fibroblasts and TGFβ1-activated myofibroblasts, respectively. The results point to dynamic interplay between transcriptional activators and repressors in regulating SMαA gene output during myofibroblast differentiation. Therapeutic targeting of nucleoprotein complexes regulating the SMαA promoter may prevent excessive myofibroblast accumulation associated with chronic cardiopulmonary fibrosis and dysfunctional tissue remodeling.

(1999). Transgenic and therapeutic targeting of smooth muscle cells and the challenges presented by phenotypic diversity. Emerging Therapeutic Targets: Vol. 3, No. 2, pp. 279-306.

Transcriptional activity of the mouse vascular smooth muscle alpha-actin gene in fibroblasts is regulated, in part, by a 30-base pair asymmetric polypurine-polypyrimidine tract containing an essential MCAT enhancer motif. The double-stranded form of this sequence serves as a binding site for a transcription enhancer factor 1-related protein while the separated single strands interact with two distinct DNA binding activities termed VACssBF1 and 2 (Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995) J. Biol. Chem. 270, 11310-11321; Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2936). VACssBF2 has been recently cloned and shown to consist of two closely related proteins, Puralpha and Purbeta (Kelm, R. J., Elder, P. K., Strauch, A. R., and Getz, M. J. (1997) J. Biol. Chem. 272, 26727-26733). In this study, we demonstrate that Puralpha and Purbeta interact with each other via highly specific protein-protein interactions and bind to the purine-rich strand of the MCAT enhancer in the form of both homo- and heteromeric complexes. Moreover, both Pur proteins interact with MSY1, a VACssBF1-like protein cloned by virtue of its affinity for the pyrimidine-rich strand of the enhancer. Interactions between Puralpha, Purbeta, and MSY1 do not require the participation of DNA. Combinatorial interactions between these three single-stranded DNA-binding proteins may be important in regulating activity of the smooth muscle alpha-actin MCAT enhancer in fibroblasts.

Transcriptional repression of the mouse vascular smooth muscle alpha-actin gene in fibroblasts and myoblasts is mediated, in part, by the interaction of two single-stranded DNA binding activities with opposite strands of an essential transcription enhancer factor-1 recognition element (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436). One of these activities, previously designated vascular actin single-stranded DNA-binding factor 2 includes two distinct polypeptides (p44 and p46) which specifically interact with the purine-rich strand of both the enhancer and a related element in a protein coding exon of the gene (Kelm, R. J., Jr., Sun, S., Strauch, A. R., and Getz, M. J. (1996) J. Biol. Chem. 271, 24278-24285). Expression screening of a mouse lung cDNA library with a vascular actin single-stranded DNA-binding factor 2 recognition element has now resulted in the isolation of two distinct cDNA clones that encode p46 and p44. One of these proteins is identical to Puralpha, a retinoblastoma-binding protein previously implicated in both transcriptional activation and DNA replication. The other is a related family member, presumably Purbeta. Comparative band shift and Southwestern blot analyses conducted with cellular p46, p44, and cloned Pur proteins synthesized in vitro and in vivo, establish identity of p46 with Puralpha and p44 with Purbeta. This study implicates Puralpha and/or Purbeta in the control of vascular smooth muscle alpha-actin gene transcription.

Background— Delineating the role of microRNAs (miRNAs) in the posttranscriptional gene regulation offers new insights into how the heart adapts to pathological stress. We developed a knockout of miR-22 in mice and investigated its function in the heart. Methods and Results— Here, we show that miR-22 –deficient mice are impaired in inotropic and lusitropic response to acute stress by dobutamine. Furthermore, the absence of miR-22 sensitized mice to cardiac decompensation and left ventricular dilation after long-term stimulation by pressure overload. Calcium transient analysis revealed reduced sarcoplasmic reticulum Ca 2+ load in association with repressed sarcoplasmic reticulum Ca 2+ ATPase activity in mutant myocytes. Genetic ablation of miR-22 also led to a decrease in cardiac expression levels for Serca2a and muscle-restricted genes encoding proteins in the vicinity of the cardiac Z disk/titin cytoskeleton. These phenotypes were attributed in part to inappropriate repression of serum response factor activity in stressed hearts. Global analysis revealed increased expression of the transcriptional/translational repressor purine-rich element binding protein B, a highly conserved miR-22 target implicated in the negative control of muscle expression. Conclusion— These data indicate that miR-22 functions as an integrator of Ca 2+ homeostasis and myofibrillar protein content during stress in the heart and shed light on the mechanisms that enhance propensity toward heart failure.

Purβ is a single-stranded nucleic acid-binding protein implicated in the injury-induced repression of genes encoding certain muscle-restricted isoforms of actin and myosin expressed in the heart, skeletal muscle, and vasculature. To better understand how the modular arrangement of the primary sequence of Purβ affects the higher order structure and function of the protein, purified recombinant Purβ was subjected to partial proteolysis in an attempt to identify a well-folded truncation protein that retained purine-rich single-stranded DNA-binding activity. Limited tryptic digestion of Purβ liberated a core ~30 kDa fragment corresponding to residues 29–305 as determined by epitope mapping and mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to self-associate while circular dichroism analysis confirmed that the Purβ core domain is stably folded in the absence of glycine-rich N- and C-terminal sequences. Comparative DNA-binding assays revealed that the isolated core domain interacts with purine-rich cis-elements from the smooth muscle α-actin gene with similar specificity but increased affinity compared to full-length Purβ. These findings suggest that the highly conserved modular repeats of Purβ fold to form a core functional domain, which mediates the specific and high affinity binding of the protein to single-stranded DNA.

Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins isolated from these two sources differ with respect to the complexity of their constituent N-linked oligosaccharide. In this study, osteonectin synthesized by bone-forming cells (osteoblasts) and platelet-producing cells (megakaryocytes) in vitro was analyzed to determine if the proteins produced were analogous in terms of glycosylation to those isolated from bone and platelets, respectively. Immunoblot analyses of osteonectin produced by the osteoblast-like cell lines, SaOS-2 and MG-63, indicated that secreted and intracellular forms of the molecule are structurally distinct. Endoglycosidase treatment and immunoblotting of osteonectin secreted from SaOS-2 and MG-63 cells, under serum-deprived conditions, suggested that the molecule possessed a complex type oligosaccharide unlike the high-mannose moiety found on bone matrix-derived osteonectin. Biosynthetic labeling of SaOS-2 cells and human megakaryocytes indicated that both cell types synthesize osteonectin de novo. Electrophoretic and glycosidase sensitivity analyses of [35S]-osteonectin isolated from lysates of metabolically labeled SaOS-2 cells and megakaryocytes indicated that these two cell types synthesize osteonectin molecules that are identical in oligosaccharide structure to the isolated bone and platelet proteins. These data suggest that the intracellular form of the osteonectin molecule is glycosylated differently in SaOS-2 cells and megakaryocytes but that the extracellular form which is secreted from platelets in vivo and osteoblasts in vitro is characterized by the presence of a complex type N-linked oligosaccharide.

Activation of the coagulation system is a characteristic feature of sickle cell anemia, which also includes clinical thrombosis. The sickle transgenic mouse abnormally expresses tissue factor (TF) on the pulmonary vein endothelium. Knowing that this aberrancy is stimulated by inflammation, we sought to determine whether nitric oxide (NO) contributes to regulation of endothelial TF expression in the sickle mouse model. We used the NY1DD sickle mouse, which exhibits a low-TF to high-TF phenotype switch on exposure to hypoxia/reoxygenation. Manipulations of NO biology, such as breathing NO or addition of arginine or L-NAME (N-nitro-L-arginine-methyl-ester) to the diet, caused significant modulations of TF expression. This was also seen in hBERK1 sickle mice, which have a different genetic background and already have high-TF even at ambient air. Study of NY1DD animals bred to overexpress endothelial nitric oxide synthase (eNOS; eNOS-Tg) or to have an eNOS knockout state (one eNOS(-/-) animal and several eNOS(+/-) animals) demonstrated that eNOS modulates endothelial TF expression in vivo by down-regulating it. Thus, the biodeficiency of NO characteristic of patients with sickle cell anemia may heighten risk for activation of the coagulation system.

Expression of the arginine/lysine transporter Cat-1 is highly induced in proliferating and stressed cells via mechanisms that include transcriptional activation. A bifunctional INE (intronic element) within the first intron of the Cat-1 gene was identified and characterized in this study. The INE had high sequence homology to an amino acid response element and was shown to act as a transcriptional enhancer in unstressed cells by binding the transcription factor, purine-rich element binding protein A (Pur alpha). During endoplasmic reticulum stress, binding of Pur alpha to the INE decreased; the element acted as a positive regulator in early stress by binding of the transcription factor ATF4 and as a negative regulator in prolonged stress by binding the stress-induced C/EBP family member, CHOP. We conclude that transcriptional control of the Cat-1 gene is tightly controlled by multiple cis-DNA elements, contributing to regulation of cationic amino acid transport for cell growth and proliferation. In addition, we propose that genes may use stress-response elements such as the INE to support basal expression in the absence of stress.

Textbook models of the transcription of eukaryotic protein-coding genes typically depict the recruitment of the RNA polymerase II machinery to a core promoter as coordinated by the pre-assembly of trans-acting factors at specific target sites in the 5′-flanking region of a gene in a manner dictated by nucleotide sequence and DNA accessibility. By-and-large, this classical view is predicated on the principle that recognition of specific DNA sequences by transcription factors involves chemical interactions between certain base-pairs oriented in the major or minor groove of duplex DNA and compatible amino acids arrayed on the surface of the protein. However, this conventional model of B-DNA-dependent activation or repression of transcription does not fully explain the regulatory intricacies of genes which harbor promoter elements that are prone to adopting non-B-DNA structures including single-stranded DNA (ssDNA)1 (1–6). Of particular interest from a thermodynamic perspective, is how sequence-specific ssDNA-binding proteins (ssBPs) participate in the formation and/or stabilization of these alternate structures to positively or negatively affect gene activity (7–12). Among the relatively small group of ssDNA-binding transcription factors indentified to date, two members of the purine-rich element (PUR) binding protein family, Purα and Purβ, have been linked to the repression of genes encoding smooth muscle α-actin (SMαA) (13), α-myosin heavy chain (14), and β-myosin heavy chain (15). Consequently, these ssBPs likely serve as downstream effectors of muscle gene reprogramming induced by physiological stress or injury in cardiac, skeletal, or vascular cell types (14–18). In the case of the SMαA gene, Purα and Purβ appear to collaborate with another ssBP known as MSY1 (mouse YB-1) to suppress the activity of a highly-conserved muscle-CAT (MCAT)-containing enhancer in growth-activated fibroblasts and vascular smooth muscle cells (13). In keeping with the general composition and properties of other ssBP recognition sites, the SMαA enhancer possesses a high level of purine-pyrimidine asymmetry (19), and lies within a region of the promoter which is hypersensitive to ssDNA-specific chemical modification (20). An ensemble of biochemical studies conducted with fibroblast-derived, smooth muscle cell-derived, and recombinant proteins have established that Purα and Purβ specifically bind to the purine-rich strand, whereas MSY1 interacts with the opposing pyrimidine-rich strand of the nominal MCAT enhancer element (13, 21). On the basis of these and other nucleoprotein binding and promoter mutation analyses, a working model for enhancer encryption was proposed envisioning concurrent binding by all three ssBPs in such a way as to inhibit MCAT-dependent activation by transcription enhancer factor 1 (13, 19). Although this model assumed equal contribution by each ssBP, more recent gain-of-function, loss-of-function, and in vivo promoter occupancy studies have suggested that Purβ may be the dominant physical and functional effector of SMαA repression in certain cell types (22–24). As a logical extension of prior work showing that 1) fibroblast-derived Purα and Purβ form deoxycholate-sensitive oligomeric complexes on the purine-rich strand of the MCAT-containing enhancer (21), 2) nucleotides near the 5′ and 3′ ends of the nominal enhancer element mediate high affinity binding by nuclear Purα and Purβ extracted from fibroblasts and vascular smooth muscle cells (13), and 3) purified recombinant Purβ reversibly self-associates in the absence of ssDNA (25), we hypothesized that dimerization may be a prerequisite to bidentate Pur protein interaction with two independent recognition sites flanking the core MCAT motif. In this study, we employed rigorous, quantitative methods to validate these putative recognition sites and to determine the mechanism of ssDNA-binding by Purβ. In contrast to obligate dimer-dependent association, our results indicate that protein binding to the purine-rich strand of the SMαA enhancer involves cooperative assembly of Purβ monomers at two slightly degenerate PUR sites to form a high affinity 2:1 protein:ssDNA complex. Moreover, the resolved energetics of strand- specific enhancer binding by Purβ suggests that formation and maintenance of an extended single-stranded state in the genomic SMαA promoter likely requires auxiliary factors, such as Purα and/or MSY1, to overcome the thermodynamic favorability of strand annealing.

Transforming growth factor (TGF) beta1 is a mediator of myofibroblast differentiation in healing wounds in which it activates transcription of the smooth muscle alpha-actin (SMalphaA) gene via dynamic interplay of nuclear activators and repressors. Targeting components of TGFbeta1 signaling may be an effective strategy for controlling myofibroblasts in chronic fibrotic diseases. We examined the ability of proinflammatory tumor necrosis factor (TNF)-alpha to antagonize TGFbeta1-mediated human pulmonary myofibroblast differentiation. TNF-alpha abrogated TGFbeta1-induced SMalphaA gene expression at the level of transcription without disrupting phosphorylation of regulatory Smads. Intact mitogen-activated protein kinase kinase (Mek)-extracellular signal-regulated kinase (Erk) kinase signaling was required for myofibroblast repression by TNF-alpha via induction of the early growth response factor-1 (Egr-1) DNA-binding protein. Egr-1 bound to the GC-rich SPUR activation element in the SMalphaA promoter and potently suppressed Smad3- and TGFbeta1-mediated transcription. Reduction in Smad binding to the SMalphaA promoter in TNF-alpha-treated myofibroblasts was accompanied by an increase in Egr-1 and YB-1 repressor binding, suggesting that the molecular mechanism underlying repression may involve competitive interplay between Egr-1, YB-1, and Smads. The ability of TNF-alpha to attenuate myofibroblast differentiation via modulation of a Mek1/Erk/Egr-1 regulatory axis may be useful in designing new therapeutic targets to offset destructive tissue remodeling in chronic fibrotic disease.

Activation of the coagulation system is a characteristic feature of sickle cell anemia, which also includes clinical thrombosis. The sickle transgenic mouse abnormally expresses tissue factor (TF) on the pulmonary vein endothelium. Knowing that this aberrancy is stimulated by inflammation, we sought to determine whether nitric oxide (NO) contributes to regulation of endothelial TF expression in the sickle mouse model. We used the NY1DD sickle mouse, which exhibits a low-TF to high-TF phenotype switch on exposure to hypoxia/reoxygenation. Manipulations of NO biology, such as breathing NO or addition of arginine or L-NAME (N-nitro-L-arginine-methyl-ester) to the diet, caused significant modulations of TF expression. This was also seen in hBERK1 sickle mice, which have a different genetic background and already have high-TF even at ambient air. Study of NY1DD animals bred to overexpress endothelial nitric oxide synthase (eNOS; eNOS-Tg) or to have an eNOS knockout state (one eNOS(-/-) animal and several eNOS(+/-) animals) demonstrated that eNOS modulates endothelial TF expression in vivo by down-regulating it. Thus, the biodeficiency of NO characteristic of patients with sickle cell anemia may heighten risk for activation of the coagulation system.

In this study we report that bone and platelet osteonectin are structurally and functionally heterogeneous in terms of glycosylation and collagen binding capacity. The relative sensitivity of bone and platelet osteonectin to specific glycosidases was used to evaluate potential differences in glycosylation. Although native bone and platelet osteonectin are electrophoretically nonidentical, N-glycanase treatment yielded products with the same apparent molecular weight. Bone osteonectin was also susceptible to cleavage by endo H but not to neuraminidase, while platelet osteonectin was susceptible to neuraminidase but not to endo H. In lectin blotting experiments of bone and platelet osteonectin, concanavalin A bound specifically to bone osteonectin but not to platelet osteonectin. However, Lens culinaris agglutinin bound to platelet osteonectin but not to bone osteonectin. These data suggest that bone and platelet osteonectin differ in their oligosaccharide side chain structures, with bone osteonectin possessing a high mannose-type and platelet osteonectin, a complex-type structure. Solid-phase binding techniques were used to functionally evaluate bone and platelet osteonectin in terms of collagen binding. Although bone osteonectin bound specifically to types I, III, and V collagen, platelet osteonectin had no apparent affinity for these collagen types suggesting that the two proteins are also functionally distinct.

Mouse hearts subjected to repeated transplant surgery and ischemia-reperfusion injury develop substantial interstitial and perivascular fibrosis that was spatially associated with dysfunctional activation of fetal smooth muscle alpha-actin (SM alpha A) gene expression in graft ventricular cardiomyocytes. Compared with cardiac fibroblasts in which nuclear levels of the Sp1 and Smad 2/3 transcriptional-activating proteins increased markedly after transplant injury, the most abundant SM alpha A gene-activating protein in cardiomyocyte nuclei was serum response factor (SRF). Additionally, cardiac intercalated discs in heart grafts contained substantial deposits of Pur alpha, an mRNA-binding protein and known negative modulator of SRF-activated SM alpha A gene transcription. Activation of fetal SM alpha A gene expression in perfusion-isolated adult cardiomyocytes was linked to elevated binding of a novel protein complex consisting of SRF and Pur alpha to a purine-rich DNA element in the SM alpha A promoter called SPUR, previously shown to be required for induction of SM alpha A gene transcription in injury-activated myofibroblasts. Increased SRF binding to SPUR DNA plus one of two nearby CArG box consensus elements was observed in SM alpha A-positive cardiomyocytes in parallel with enhanced Pur alpha:SPUR protein:protein interaction. The data suggest that de novo activation of the normally silent SM alpha A gene in reprogrammed adult cardiomyocytes is linked to elevated interaction of SRF with fetal-specific CArG and injury-activated SPUR elements in the SM alpha A promoter as well as the appearance of novel Pur alpha protein complexes in both the nuclear and cytosolic compartments of these cells.

Our laboratory has previously shown that osteonectin, an abundant noncollagenous bone protein, is contained in and secreted from human platelets. In this study, the distribution of osteonectin both in the supernatant and on the platelet surface after activation was measured by fluid-phase and solid-phase radioimmunoassay, respectively. Total cellular osteonectin was determined by RIA of guanidinium chloride extracted platelets and ranged from 0.65 to 2.2 micrograms/10(8) platelets or 135,000 to 457,000 molecules/platelet. Platelets treated with varying concentrations of collagen and thrombin released osteonectin in a dose-dependent fashion. Approximately 61% of the total platelet osteonectin was secreted at saturating concentrations of collagen and thrombin. A small fraction of platelet osteonectin is expressed on the surface of platelets in an activation-specific manner as evidenced by the specific and saturable binding of [125I]-anti- osteonectin monoclonal antibody, IIIA3A8, to thrombin-activated platelets. Based on a non-linear least squares regression analysis of the antibody binding, 2,200 IIIA3A8 molecules, or 0.8% of the total platelet osteonectin, is expressed on the platelet surface on activation. Platelet osteonectin was purified from the supernatant of thrombin-activated platelets by immunoaffinity chromatography. Western blotting of proteins secreted by washed, thrombin-stimulated platelets with IIIA3A8 indicated that the osteonectin molecule released from the platelet is a single chain polypeptide. Comparison of immunopurified platelet osteonectin with isolated bovine bone osteonectin and isolated human bone osteonectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that platelet osteonectin has a greater apparent molecular weight than bone osteonectin. The NH2-terminal sequence of immunopurified platelet osteonectin was obtained by automated Edman degradation and is identical to the sequence of human bone osteonectin derived from the cDNA of SaOS-2 cells. Collectively, these data suggest that platelet osteonectin is structurally distinct from bone osteonectin in a region of the molecule at a distance from the NH2-terminus.

Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins, Pur alpha and Pur beta, in transcriptional repression of the smooth muscle alpha-actin gene in vascular cell types. Although Pur alpha and Pur beta share substantial sequence homology and nucleic acid binding properties, genomic promoter and cis-element occupancy studies reported herein suggest that Pur beta is the dominant factor in gene regulation. To dissect the molecular basis of Pur beta repressor activity, site-directed mutagenesis was used to map amino acids critical to the physical and functional interaction of Pur beta with the smooth muscle alpha-actin promoter. Of all the various acidic, basic, and aromatic residues studied, mutation of positionally conserved arginines in the class I or class II repeat modules significantly attenuated Pur beta repressor activity in transfected vascular smooth muscle cells and fibroblasts. DNA binding and protein-protein interaction assays were conducted with purified recombinant Pur beta and selected mutants to reveal the physical basis for loss-of-function. Mutants R57E, R57E/R96E, and R57A/R96A each exhibited reduced single-stranded DNA binding affinity for an essential promoter element and diminished interaction with corepressor YB-1/MSY1. Structural analyses of the R57A/R96A and R57E/R96E double mutants in comparison to the wild-type Pur beta homodimer revealed aberrant self-association into higher order oligomeric complexes, which correlated with decreased alpha-helical content and defective DNA and protein binding in vitro. These findings point to a previously unrecognized structural role for certain core arginine residues in forming a conformationally stable Pur beta protein capable of physical interactions necessary for smooth muscle alpha-actin gene repression.

Clinically, the vascular pathobiology of human sickle cell disease includes an abnormal state of chronic inflammation and activation of the coagulation system. Since these biologies likely underlie development of vascular disease in sickle subjects, they offer attractive targets for novel therapeutics. Similar findings characterize the sickle transgenic mouse, which therefore provides a clinically relevant inflammation model.The authors tested two polyhydroxyphenyl hydroxamic acid derivatives, didox and trimidox, in sickle transgenic mice. Animals were examined by intravital microscopy (cremaster muscle and dorsal skin fold preparations) and by histochemistry before and after transient exposure to hypoxia, with versus without preadministration of study drug. Previous studies have validated the application of hypoxia/reoxygenation to sickle transgenic mice as a disease-relevant model.Animals pretreated with these agents exhibited marked improvements in leukocyte/ endothelial interaction, hemodynamics and vascular stasis, and endothelial tissue factor expression. Thus, these drugs unexpectedly exert powerful inhibition on both the inflammation and coagulation systems.Each of these changes is expected to be therapeutically beneficial in systemic inflammatory disease in general, and in sickle disease in particular. Thus, these novel compounds offer the advantage of having multiple therapeutic benefits in a single agent.

Pur alpha and Pur beta are structurally related single-stranded DNA/RNA-binding proteins implicated in the control of cell growth and differentiation. The goal of this study was to determine whether Pur alpha and Pur beta function in a redundant, distinct, or collaborative manner to suppress smooth muscle alpha-actin gene expression in cell types relevant to wound repair and vascular remodeling. RNA interference-mediated loss-of-function analyses revealed that, although Pur beta was the dominant repressor, the combined action of endogenous Pur alpha and Pur beta was necessary to fully repress the full-length smooth muscle alpha-actin promoter in cultured fibroblasts but to a lesser extent in vascular smooth muscle cells. The activity of a minimal core enhancer containing a truncated 5' Pur repressor binding site was unaffected by knockdown of Pur alpha and/or Pur beta in fibroblasts. Conversely, gain-of-function studies indicated that Pur alpha or Pur beta could each independently repress core smooth muscle alpha-actin enhancer activity albeit in a cell type-dependent fashion. Biochemical analyses indicated that purified recombinant Pur alpha and Pur beta were essentially identical in terms of their binding affinity and specificity for GGN repeat-containing strands of several cis-elements comprising the core enhancer. However, Pur alpha and Pur beta exhibited more distinctive protein interaction profiles when evaluated for binding to enhancer-associated transcription factors in extracts from fibroblasts and vascular smooth muscle cells. These findings support the hypothesis that Pur alpha and Pur beta repress smooth muscle alpha-actin gene transcription by means of DNA strand-selective cis-element binding and cell type-dependent protein-protein interactions.

The rat aldolase B promoter acts as a replication origin in vivo, as well as an autonomously replicating sequence (ARS). Here, we examined roles of a polypurine stretch (site PPu) in this origin, which is indispensable to the ARS activity. Purification of site PPu-binding protein revealed that site PPu binds Puralpha and Purbeta, i.e., single-stranded DNA-binding proteins whose roles in replication have been implicated, but less clear. Biochemical analyses showed that site PPu even in a longer DNA fragment is unstable in terms of double-helix, implying that Puralpha/beta may stabilize single-stranded state. Deletion of site PPu from the origin DNA, which was ectopically positioned in the mouse chromosome, significantly reduced replicator activity. Chromatin immunoprecipitation experiments showed that deletion of site PPu abolishes binding of the Puralpha/beta proteins to the origin. These observations suggest functional roles of site PPu and Puralpha/beta proteins in replication initiation.

Expression of smooth muscle alpha-actin in growth factor-induced myofibroblasts and in differentiated vascular smooth muscle cells is transcriptionally controlled by multiple positive or negative trans-acting factors interacting with distinct cis-elements in the 5'-flanking region of the gene. Because none of the transcriptional regulators reported to date is smooth muscle cell- or myofibroblast-specific per se, the dynamic interplay among many factors interacting at specific sites along the promoter appears to be a signature feature of smooth muscle alpha-actin gene regulation in these cell types. Herein, the ability of the adenovirus E1A 12 S protein to bind and functionally inactivate specific cell regulatory factors has been exploited to identify several previously unknown coactivators of the mouse smooth muscle alpha-actin promoter in rodent fibroblasts and vascular smooth muscle cells. In transient cotransfection assays, ectopic expression of wild type E1A suppressed promoter activity in a dose- and cis-element-dependent manner. In asynchronous cells, N-terminal E1A mutants defective in CREB-binding protein (CBP) and p300 binding capacity exhibited markedly reduced inhibitory activity toward a smooth muscle alpha-actin promoter driven by a composite TEF-1-, SRF-, and Sp1/3-regulated enhancer. In synchronized cells, however, a more complex mutant E1A inhibitory pattern indicated that collaboration between CBP/p300 and the retinoblastoma family of pocket proteins was required to produce a fully functional enhancer. Cotransfection experiments conducted with Rb(-/-) fibroblasts demonstrated the necessity of pRB in augmenting smooth muscle alpha-actin enhancer/promoter activity. Physical interaction studies with the use of purified wild type and mutant E1A proteins confirmed that CBP, p300, and pRB were targets of E1A binding in nuclear extracts of vascular smooth muscle cells and/or fibroblasts. Collectively, these results suggest that a repertoire of E1A-interacting proteins, namely CBP/p300 and pRB, serve to integrate the activities of multiple trans-acting factors to control smooth muscle alpha-actin gene transcription in a cell type- and cell cycle-dependent manner.

The mouse vascular smooth muscle alpha-actin (SMA) gene enhancer is activated in fibroblasts by transforming growth factor beta1 (TGFbeta1), a potent mediator of myofibroblast differentiation and wound healing. The SMA enhancer contains tandem sites for the Sp1 transcriptional activator protein and Puralpha and beta repressor proteins. We have examined dynamic interplay between these divergent proteins to identify checkpoints for possible control of myofibroblast differentiation during chronic inflammatory disease. A novel element in the SMA enhancer named SPUR was responsible for both basal and TGFbeta1-dependent transcriptional activation in fibroblasts and capable of binding Sp1 and Pur proteins. A novel Sp1:Pur:SPUR complex was dissociated when SMA enhancer activity was increased by TGFbeta1 or Smad protein overexpression. Physical association of Pur proteins with Smad2/3 was observed as was binding of Smads to an upstream enhancer region that undergoes DNA duplex unwinding in TGFbeta1-activated myofibroblasts. Purbeta repression of the SMA enhancer could not be relieved by TGFbeta1, whereas repression mediated by Puralpha was partially rescued by TGFbeta1 or overexpression of Smad proteins. Interplay between Pur repressor isoforms and Sp1 and Smad coactivators may regulate SMA enhancer output in TGFbeta1-activated myofibroblasts during episodes of wound repair and tissue remodeling.

Increased expression of plasminogen activator inhibitor type 1 (PAI-1) is associated with decreased apoptosis of neoplastic cells. We sought to determine whether PAI-1 alters apoptosis in vascular smooth muscle cells (VSMC) and, if so, by what mechanisms. A twofold increase in the expression of PAI-1 was induced in VSMC from transgenic mice with the use of the SM-22alpha gene promoter (SM22-PAI+). Cultured VSMC from SM22-PAI+ mice were more resistant to apoptosis induced by tumor necrosis factor plus phorbol myristate acetate or palmitic acid compared with VSMC from negative control littermates. Both wild type (WT) and a stable active mutant form of PAI-1 (Active) inhibited caspase-3 amidolytic activity in cell lysates while a serpin-defective mutant (Mut) PAI-1 did not. Similarly, both WT and Active PAI-1 decreased amidolytic activity of purified caspase-3, whereas Mut PAI-1 did not. WT but not Mut PAI-1 decreased the cleavage of poly-[ADP-ribose]-polymerase (PARP), the physiological substrate of caspase-3. Noncovalent physical interaction between caspase-3 and PAI-1 was demonstrable with the use of both qualitative and quantitative in vitro binding assays. High affinity binding was eliminated by mutations that block PAI-1 serpin activity. Accordingly, attenuated apoptosis resulting from elevated expression of PAI-1 by VSMC may be attributable, at least in part, to reversible inhibition of caspase-3 by active PAI-1.

Abnormal tissue factor (TF) expression has been demonstrated on blood monocytes and circulating endothelial cells in humans with sickle cell anemia. We have now studied sickle transgenic mice to help define the biology of endothelial TF expression in sickle disease. Using immunostaining of tissue sections, we find that this is confined almost exclusively to the pulmonary veins. About 15% and 13% of these exhibit TF-positive endothelium in the wild-type normal mouse and the normal human hemoglobin (HbA)-expressing control transgenic mouse, respectively. The mild sickle mouse is indistinguishable from normal (approximately 14% positive), but TF expression is significantly elevated in the moderate and severe mouse models of sickle disease (approximately 29% and approximately 41% positive, respectively). Exposure of the mild sickle mouse to hypoxia for 3 hours, followed by reoxygenation, converted its TF expression phenotype to that of the severe sickle mouse (approximately 36% positive). Pretreatment with lovastatin eliminated excessive expression of TF in the posthypoxic mild sickle mouse (approximately 16% positive) and in the more severe mouse at ambient air (approximately 21% positive). In addition to identifying tissue expression of endothelial TF in the sickle lung, these studies implicate reperfusion injury physiology in its expression and suggest a rationale for use of statins in sickle disease.

The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor beta1 (TGF-beta1) and accompanied by transient activation of the vascular smooth muscle alpha-actin (SmalphaA) gene. Multiple positive-regulatory elements were identified as essential mediators of basal SmalphaA enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-beta1. However, TGF-beta1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the SmalphaA enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate SmalphaA enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the SmalphaA enhancer required for TGF-beta1 amplification. Additionally, the TGF-beta1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for SmalphaA enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the SmalphaA enhancer. The identification of Sp and Smad proteins as essential, independent activators of the SmalphaA enhancer provides new insight into the poorly understood process of myofibroblast differentiation.