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Protein kinase C (PKC) isoforms are critically involved in the regulation of innate immune responses. Herein, we investigated the role of conventional PKCalpha in the regulation of IFN-beta gene expression mediated by the Toll-like receptor 3 (TLR3) signaling pathway. Inhibition of conventional PKC (cPKC) activity in monocyte-derived dendritic cells or TLR3-expressing cells by an isoform-specific inhibitor, Gö6976, selectively inhibited IFN-beta synthesis induced by double-stranded RNA polyinosine-polycytidylic acid. Furthermore, reporter gene assays confirmed that PKCalpha regulates IFN-beta promoter activity, since overexpression of dominant negative PKCalpha but not PKCbeta(I) repressed interferon regulatory factor 3 (IRF-3)-dependent but not NF-kappaB-mediated promoter activity upon TLR3 engagement in HEK 293 cells. Dominant negative PKCalpha inhibited IRF-3 transcriptional activity mediated by overexpression of TIR domain-containing adapter inducing IFN-beta and Tank-binding kinase-1. Additional biochemical analysis demonstrated that Gö6976-treated dendritic cells exhibited IRF-3 phosphorylation, dimerization, nuclear translocation, and DNA binding activity analogous to their control counterparts in response to polyinosine-polycytidylic acid. In contrast, co-immunoprecipitation experiments revealed that TLR3-induced cPKC activity is essential for mediating the interaction of IRF-3 but not p65/RelA with the co-activator CREB-binding protein. Furthermore, PKCalpha knock-down with specific small interfering RNA inhibited IFN-beta expression and down-regulated IRF-3-dependent promoter activity, establishing PKCalpha as a component of TLR3 signaling that regulates IFN-beta gene expression by targeting IRF-3-CREB-binding protein interaction. Finally, we analyzed the involvement of cPKCs in other signaling pathways leading to IFN-beta synthesis. These experiments revealed that cPKCs play a role in the synthesis of IFN-beta induced via both TLR-dependent and -independent pathways.

Considerable research has focused on the anti-inflammatory and antiproliferative activities exhibited by the soy isoflavone genistein. We previously demonstrated that genistein suppresses TNF-α-induced NF-κB-dependent IL-6 gene expression in cancer cells by interfering with the mitogen- and stress-activated protein kinase 1 activation pathway. However, effects of isoflavones on immune cells, such as dendritic cells, remain largely unknown. Here we show that genistein markedly reduces IL-6 cytokine production and transcription in LPS-stimulated human monocyte-derived dendritic cells. More particularly, we observe that genistein inhibits IL-6 gene expression by modulating the transcription factor NF-κB. Examination of NF-κB-related events downstream of TLR4 demonstrates that genistein affects NF-κB subcellular localization and DNA binding, although we observe only a minor inhibitory impact of genistein on the classical LPS-induced signaling steps. Interestingly, we find that genistein significantly increases p53 protein levels. We also show that overexpression of p53 in TLR4/MD2 HEK293T cells blocks LPS-induced NF-κB-dependent gene transcription, indicating the occurrence of functional cross-talk between p53 and NF-κB. Moreover, analysis of IL-6 mRNA levels in bone marrow-derived p53 null vs wild-type dendritic cells confirms a role for p53 in the reduction of NF-κB-dependent gene expression, mediated by genistein.

The synthesis of interferon-β (IFNβ) and IFN-inducible factors elicited by lipopolysaccharide (LPS) depends on the transcriptional activity of interferon regulatory factor 3 (IRF-3) downstream of Toll-like receptor-4 (TLR4). To examine the ability of human newborns to mount TLR4-mediated IRF-3–dependent responses, we analyzed the pattern of genes expressed on the addition of LPS to cord blood or cord blood monocyte-derived dendritic cells (moDCs). Expression of IFNβ and IFN-inducible genes was selectively impaired in neonatal blood and moDCs as compared with their adult counterparts. This selective defect was confirmed by microarray experiments on moDCs. Altered expression of IFN-inducible genes was related to impaired IFNβ synthesis because IFNβ signaling was functional in neonatal moDCs. However, addition of exogenous IFNβ failed to restore LPS-induced IL-12p70 synthesis which was previously shown to be defective in neonatal moDCs. Although LPS-induced IRF-3 nuclear translocation was observed both in adult and neonatal moDCs, IRF-3 DNA-binding activity and association with the coactivator CREB-binding protein (CBP) were decreased in neonatal as compared with adult moDCs. We conclude that impaired IRF-3/CBP interaction in neonatal blood cells exposed to LPS is associated with impaired expression of IFNβ and IFN-inducible genes. Because IRF-3 activity is also required for IL-12p70 synthesis, our findings provide a molecular basis for the decreased ability of LPS-stimulated neonatal moDCs to elicit Th1-type responses.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by dendritic cells (DCs) in response to Toll-like receptor (TLR) ligation. While the mechanisms regulating IL-12p40 chain gene expression are well characterized, molecular events involved in IL-12p35 chain gene activation remain to be clarified. Since IL-12p35 mRNA was induced in human DCs activated through TLR3 or TLR4 but not TLR2, we investigated the potential role of interferon regulatory factor 3 (IRF-3) in IL-12p35 gene transactivation. First, a binding site for IRF-3 named interferon-stimulated response element-1 (ISRE-1) was identified in the human IL-12p35 promoter region between nucleotides -251 and -240. The ISRE-1 site was required for IL-12p35 gene activation in RAW 264.7 cells stimulated by lipopolysaccharide (LPS) or PolyI:C. Ectopic expression of IRF-3 was found to up-regulate IL-12p35 gene activation in the same system. Furthermore, chromatin immunoprecipitation (ChIP) studies demonstrated that IRF-3 is recruited to ISRE-1 site in TLR4- or TLR3-stimulated human DCs. Finally, experiments on DCs from IRF-3-deficient mice established that TLR4-induced IL-12p35 mRNA and IL-12p70 synthesis are impaired in absence of IRF-3. We conclude that IRF-3 binds to a critical cis-acting element in the IL-12p35 gene promoter and thereby represents a key factor for the induction of IL-12p70 synthesis in DCs.

Whereas the tumor acidic extracellular pH plays a crucial role in the invasive process, the mechanism(s) behind this acidification, especially in low nutrient conditions, are unclear. The regulation of the Na(+)/H(+) exchanger (NHE) and invasion by serum deprivation were studied in a series of breast epithelial cell lines representing progression from non-tumor to highly metastatic cells. Whereas serum deprivation reduced lactate production in all three cells lines, it inhibited NHE activity in the non-tumor cells and stimulated it in the tumor cells with a larger stimulation in the metastatic cells. The stimulation of NHE in the tumor cell lines was the result of an increased affinity of the internal H(+) regulatory site of the NHE without changes in sodium kinetics or expression. Serum deprivation conferred increased cell motility and invasive ability that were abrogated by specific inhibition of the NHE. Inhibition of phosphoinositide 3-kinase by overexpression of a dominant-negative mutant or wortmannin incubation inhibited NHE activity and invasion in serum replete conditions while potentiating the serum deprivation-dependent activation of the NHE and invasion. These results indicate that the up-regulation of the NHE by a phosphoinositide 3-kinase-dependent mechanism plays an essential role in increased tumor cell invasion induced by serum deprivation.

The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homodimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration.

Nonunion fractures can cause severe dysfunction and are often difficult to treat mainly due to a poor understanding of their physiopathology. Although many aspects of impaired fracture healing have been extensively studied, little is known about the cellular and molecular mechanisms leading to atrophic nonunion. Therefore, the aim of the present study was to assess the pools and biological functions of bone marrow-derived mesenchymal stem cells (hMSCs) and circulating endothelial progenitor cells (EPCs) in atrophic nonunion patients compared to healthy subjects, and the systemic levels of growth factors involved in the recruitment, proliferation and differentiation of these cells. In nonunions, the pool of hMSCs was decreased and their proliferation delayed. However, once committed, hMSCs from nonunions were able to proliferate, differentiate into osteoblastic cells and mineralize in vitro as efficiently as hMSCs from healthy subjects. In parallel, we found altered serum levels of chemokines and growth factors involved in the chemotaxis and proliferation of hMSCs such as leptin, interleukin-6 (IL-6) and its soluble receptor, platelet-derived growth factor-BB (PDGF-BB), stem cell factor (SCF) and insulin-like growth factor-1 (IGF-1). Moreover, we showed that the number of EPCs and their regulating growth factors were not affected in nonunion patients. If nonunion is generally attributed to a vascular defect, our results also support a role for a systemic mesenchymal and osteogenic cell pool defect that might be related to alterations in systemic levels of factors implicated in their chemotaxis and proliferation.

In order to verify whether the HER-2/neu gene is involved in the initial phases of neoplastic disease or in its progression, we evaluated the amplification and overexpression of this gene in the primary tumor and in synchronous metastatic axillary lymph nodes of 26 women with operable breast cancer. HER-2/neu was amplified in 35% and overexpressed in 33% of the primary sites; similar percentages were found in lymph nodes. The clear correlation between the two disease sites regarding gene, mRNA and protein levels, supports the hypothesis that this gene is involved in the initial and invasive phases of neoplasia. Its actual role with respect to other biological tumor characteristics during the metastatic process should be investigated further.

Quiescent, serum-starved human aortic smooth muscle cells were restimulated with 20% foetal calf serum in Dulbecco's modified Eagle medium, in the presence and absence of beta-carotene, canthaxanthin, zeaxanthin, lycopene, lutein or beta-cryptoxanthin, at final concentrations up to 23 microM. Concentration-dependent inhibition of DNA synthesis, measured by [methyl-3H]thymidine incorporation, was observed for the carotenoids, except for canthaxanthin and lutein which had no effect. Lycopene was the most potent of the carotenoids tested. The results suggest that antiproliferative effects of dietary carotenoids might be of significance in vivo.

In this study we investigate the mechanism of intracellular pH change and its role in malignant transformation using the E7 oncogene of human papillomavirus type 16 (HPV16). Infecting NIH3T3 cells with recombinant retroviruses expressing the HPV16 E7 or a transformation deficient mutant we show that alkalinization is transformation specific. In NIH3T3 cells in which transformation can be turned on and followed by induction of the HPV16 E7 oncogene expression, we demonstrate that cytoplasmic alkalinization is an early event and was driven by stimulation of Na+/H+ exchanger activity via an increase in the affinity of the intracellular NHE-1 proton regulatory site. Annulment of the E7-induced cytoplasmic alkalinization by specific inhibition of the NHE-1, acidification of culture medium, or clamping the pHi to nontransformed levels prevented the development of later transformed phenotypes such as increased growth rate, serum-independent growth, anchorage-independent growth, and glycolytic metabolism. These findings were verified in human keratinocytes (HPKIA), the natural host of HPV. Results from both NIH3T3 and HPKIA cells show that alkalinization acts on pathways that are independent of the E2F-mediated transcriptional activation of cell cycle regulator genes. Moreover, we show that the transformation-dependent increase in proliferation is independent of the concomitant stimulation of glycolysis. Finally, treatment of nude mice with the specific inhibitor of NHE-1, DMA, delayed the development of HPV16-keratinocyte tumors. Our data confirm that activation of the NHE-1 and resulting cellular alkalinization is a key mechanism in oncogenic transformation and is necessary for the development and maintenance of the transformed phenotype.

The present study evaluated the presence of GnRH-R in leiomyomas, in associated, non-involved uterine tissues (myometrium and endometrium) and the possible relationships between GnRH-R and the receptors for estrogen and progesterone in the same tissues. GnRH-R was found in all uterine tissues and both GnRH and the GnRH analog, goserelin, displaced its binding consistent with a single type of high affinity receptor (Kd approximate to 10(-8) M). GnRH-R were found more frequently in myometrium (81% of samples) than in endometrium (58%) or leiomyoma (42%). However, the mean receptor content was lowest in myometrium (139+/-19 fmol/mg protein) with both leiomyomas (288+/-77 fmol/mg protein) and endometrium (372+/-96 fmol/mg protein) having significantly higher values. Endometrial GnRH binding varied from 596+/-42 in uteri that were GnRH-R positive in the endothelium alone to 231+/-49 when GnRH-R was present also in the other tissues. Endometrium negative for the GnRH-R had significantly higher levels of estrogen receptor than all the other uterine samples (266+/-25 vs 61+/-7.5 fmol/mg protein, respectively). Endometrial GnRH-R seem to be dependent on its presence and/or level in other uterine tissues. Further, when GnRH-R is absent in the endometrium this tissue expresses greatly increased levels of steroid receptors.

The peptide hormone, GnRH has been hypothesized to play a direct antitumoral role in certain kinds of breast cancer. This direct effect is considered to function via high affinity membrane receptors to GnRH that have been demonstrated in tumors. However, no routine assay for GnRH receptors exist limiting further research in elucidating the mechanisms of GnRH action during treatment and both the clinical usefulness of this measure and the development or refinement of GnRH treatments. Part of the problem has been that previously reported procedures require expensive, difficult (for routine clinical laboratories) iodination of specific GnRH analogs. The use of iodinated substrates present the additional problems of radioactive waste disposal and safety of laboratory personal. Further, the use of different analogs complicates the comparison of data between laboratories. We present an assay that utilizes tritiated natural GnRH in order to assay the native receptor binding and reduce both safety problems and cost of assay/waste disposal. The use of nitrocellulose filters treated overnight with 1% BSA resolved the problems of high blank values. Kinetic data demonstrate that this assay is sensitive, accurate and give results comparable with other methods. We also show that the membranes derived from the tumor and the cell cultures may be frozen at -80 degrees C for up to 90 days without affecting the results of the assay.