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Activating transcription factor 4 (ATF4) is a member of the basic leucine zipper (bZip) transcription factor family required for the terminal differentiation of osteoblasts. Despite its critical importance as one of the three main osteoblast differentiation transcription factors, regulators of osteoblast terminal maturation remain poorly defined. Here we report the identification of homologous pairing protein 2 (Hop2) as a dimerization partner of ATF4 in osteoblasts via the yeast two-hybrid system. Deletional mapping revealed that the Zip domain of Hop2 is necessary and sufficient to bind ATF4 and to enhance ATF4-dependent transcription. Ectopic Hop2 expression in preosteoblasts increased endogenous ATF4 protein content and accelerated osteoblast differentiation. Mice lacking Hop2 (Hop2-/- ) have a normal stature but exhibit an osteopenic phenotype similar to the one observed in Atf4-/- mice, albeit milder, which is associated with decreased Osteocalcin mRNA expression and reduced type I collagen synthesis. Compound heterozygous mice (Atf4+/- :Hop2+/- ) display identical skeletal defects to those found in Hop2-/- mice. These results indicate that Hop2 plays a previous unknown role as a determinant of osteoblast maturation via its regulation of ATF4 transcriptional activity. Our work for the first time reveals a function of Hop2 beyond its role in guiding the alignment of homologous chromosomes. © 2019 American Society for Bone and Mineral Research.

Arterial calcification predicts accelerated restenosis after angioplasty and stenting. We studied the effects of calcification on neointimal hyperplasia after balloon injury in the rat carotid. Arterial calcification was induced by subcutaneous injection of vitamin D3 or by adventitial application of calcium chloride. After balloon catheter injury, neointimal hyperplasia was significantly increased in rats with medial calcification compared with controls. Neointimal cell proliferation in calcified arteries as assessed by proliferating cell nuclear antigen (PCNA) staining was also higher. In calcified arteries, bone morphogenetic protein 2 (BMP-2)levels were increased at the time of injury suggesting a possible explanation for the altered responses. In vascular smooth muscle cells (SMCs) grown under calcifying conditions , stimulation with BMP-2 significantly increased cell proliferation, however, this did not occur in those grown under non-calcifying conditions. These data suggest that neointimal hyperplasia is accelerated in calcified arteries and that this may be due in part to increased BMP-2 expression in medial SMCs. Treatments aimed at inhibiting restenosis in calcified arteries may differ from those that work in uncalcified vessels.

Vascular calcification is a pathologic response to mineral imbalances and is prevalent in atherosclerosis, diabetes mellitus, and chronic kidney disease. When located in the media, it is highly associated with increased cardiovascular morbidity and mortality, particularly in patients on dialysis. Vascular calcification is tightly regulated and controlled by a series of endogenous factors. In the present study, we assess the effects of lysosomal and endosomal inhibition on calcification in vascular smooth muscle cells (VSMCs) and aortic rings. We observed that lysosomal function was increased in VSMCs cultured in calcification medium containing 3.5 mM inorganic phosphate (Pi) and 3 mM calcium (Ca2+) for 7 days. We also found that the lysosomal marker lysosome-associated membrane protein 2 was markedly increased and colocalized with osteogenic markers in calcified aortas from vitamin D3-treated rats. Interestingly, both the lysosomal inhibitor chloroquine and the endosomal inhibitor dynasore dose-dependently enhanced Pi + Ca2+-mediated VSMC calcification. Inhibition of lysosomal and endosomal function also promoted osteogenic transformation of VSMCs. Additionally, lysosome inhibition increased Pi-induced medial calcification of aortic rings ex vivo. These data suggest that the endosome-lysosome system may play a protective role in VSMC and medial artery calcification.

CCAAT enhancer binding protein (CEBP) transcription factors (TFs) are known to promote adipocyte differentiation; however, suppressors of CEBP TFs have not been reported thus far. Here, we find that homologous chromosome pairing protein 2 (Hop2) functions as an inhibitor for the TF CEBPα. We found that Hop2 mRNA is highly and specifically expressed in adipose tissue, and that ectopic Hop2 expression suppresses reporter activity induced by CEBP as revealed by DNA transfection. Recombinant and ectopically expressed Hop2 was shown to interact with CEBPα in pull-down and coimmunoprecipitation assays, and interaction between endogenous Hop2 and CEBPα was observed in the nuclei of 3T3 preadipocytes and adipocytes by immunofluorescence and coimmunoprecipitation of nuclear extracts. In addition, Hop2 stable overexpression in 3T3 preadipocytes inhibited adipocyte differentiation and adipocyte marker gene expression. These in vitro data suggest that Hop2 inhibits adipogenesis by suppressing CEBP-mediated transactivation. Consistent with a negative role for Hop2 in adipogenesis, ablation of Hop2 (Hop2−/−) in mice led to increased body weight, adipose volume, adipocyte size, and adipogenic marker gene expression. Adipogenic differentiation of isolated adipose-derived mesenchymal stem cells showed a greater number of lipid droplet–containing colonies formed in Hop2−/− adipose-derived mesenchymal stem cell cultures than in wt controls, which is associated with the increased expression of adipogenic marker genes. Finally, chromatin immunoprecipitation revealed a higher binding activity of endogenous CEBPα to peroxisome proliferator–activated receptor γ, a master adipogenic TF, and a known CEBPα target gene. Therefore, our study identifies for the first time that Hop2 is an intrinsic suppressor of CEBPα and thus adipogenesis in adipocytes.

Vascular calcification is highly prevalent in patients with diabetes mellitus and chronic kidney disease. When located in the media, arterial calcification is strongly associated with increased cardiovascular morbidity and mortality. The second messenger cyclic nucleotides cAMP and cGMP, controlled by distinct cyclic nucleotide phosphodiesterase (PDE) isozymes, play important regulatory roles in a variety of human diseases. Using a qPCR PDE array, we found that PDE10A was the most highly induced among all PDE genes in a rat model of medial artery calcification. PDE10A expression was markedly increased in calcified arteries from rats with chronic kidney disease and in tibial arteries from patients with peripheral artery disease. Interestingly, it co-localized with osteogenic markers in these specimens. In vitro , PDE10A knockdown using siRNA, and inhibition with a synthetic inhibitor markedly reduced osteogenic transformation and calcification of vascular SMC exposed to high phosphate levels. Aortic rings from PDE10A knockout mice showed significantly less Pi-induced medial calcification than those from wild-type controls. Deficiency of PDE10A also reduced medial calcification in a mouse medial calcification model in vivo . Mechanistic studies to elucidate the signaling alterations invoked by PDE10A are ongoing. These findings suggest that PDE10A plays a crucial role in the development of medial artery calcification, and that targeting it may provide a novel therapeutic strategy for reducing medial calcification and improving outcomes in patients with PAD.

Objective: Medial artery calcification is associated with increased cardiovascular morbidity and mortality. It occurs with diabetes and chronic kidney disease. We have previously demonstrated that the matrix metallopeptidase 3 (MMP-3) is strongly induced in arterial calcification. We also showed that MMP-3 inhibition decreases calcium accumulation in vascular SMCs and that MMP-3 deficient mice develop less medial calcification than wildtype controls. In this series of continuing experiments, we evaluate the effects of MMP-3 on SMCs phenotypic transformation in vitro and in vivo. Methods and Results: Confluent rat aortic smooth muscle cells (RASMCs) cultured in calcification medium containing elevated calcium and phosphate levels for 7 days showed increased MMP-3 activity, decreased expression of the SMC markers SM-actin and SM-MHC, and increased expression of the bone markers alkaline phosphatase (ALP) and osterix (Osx). Cells were next exposed a selective MMP-3 inhibitor 2(EMD Millipore). In the presence of inhibitor, MMP-3 activity (MMP-3 activity assay kit, Abcam) was significantly decreased. Additionally, SMC osteogenic transformation was prevented as demonstrated by maintenance of SM22α and SM-MHC expression with reduction of ALP and Osx expression. In confirmatory experiments, MMP-3 reduction with siRNA inhibited the calcification of SMC that were exposed to calcification medium. We next evaluated phenotypic marker expression in MMP-3 knockout and wild-type mice injected with vitamin D 3 , a model of medial artery calcification. At 7 days after injection, expression of the SMC marker gene SM-MHC was significantly greater in MMP-3 KO mice than controls, while bone cell marker genes (Runx2, ALP, Osx) were decreased as measured by qPCR. Deletion of MMP-3 thus inhibited the osteogenic transformation of medial SMCs in vitamin D 3 -treated mice suggesting that it may control calcification via local effects within the arterial wall. Conclusion: Together these findings suggest that MMP-3 promotes medial artery calcification through local effects on the phenotypic state of vascular SMCs, and further, that it may serve as a therapeutic target to reduce calcification and improve outcomes in our PAD patient population.

Objectives: Clinical studies demonstrate associations between arterial calcification and adverse outcomes in patients with peripheral arterial disease. The causal relationship between calcification and ischemia, however, remains unclear. This study aims to evaluate the effects of arterial calcification on functional recovery and limb perfusion following acute limb ischemia in a murine model. Methods: Arterial calcification was induced in 64 Sprague-Dawley rats using Vitamin D 3 . A control group of 32 rats received vehicle only. After 2 weeks, to allow for calcification, unilateral hindlimb ischemia was induced by ligation and excision of the superficial femoral artery. At weekly intervals, functional and ischemia status were evaluated in a blinded manner and laser Doppler perfusion imaging was performed. Results: Calcium assay demonstrated greater arterial calcification in the superficial femoral arteries (0.1 vs. 17.0, P < .01) of Vitamin D 3 injected animals. Rats injected with Vitamin D3 showed significantly worse Tarlov, Ischemia, Modified Ischemia, and Total Ischemia scores at each post-operative time point (Week 1: 16.6 vs. 14.5 P Conclusions: Vitamin D3 induced calcification is associated with decreased perfusion and worse functional recovery in a rat model of hindlimb ischemia. Future research aimed at understanding the relationship between vitamin D 3 , arterial calcification, and lower extremity ischemia may help to improve outcomes in patients with PAD.

Objective: Medial artery calcification is associated with increased cardiovascular morbidity and mortality in patients with peripheral artery disease (PAD). It is highly prevalent in diabetes and chronic kidney disease, and it is an important risk factor for cardiovascular events. Accumulating evidences suggest that matrix degrading enzymes are prominently involved in this process. Clinical studies have shown that the matrix metallopeptidase3 (MMP-3, stromelysin-1) is correlated with prevalent arterial calcification in high risk human populations. In this study, we sought to determine whether reducing MMP-3 activity could decrease arterial calcification in smooth muscle cells, organ culture, and in vivo models. Methods and Results: Confluent human aortic smooth muscle cells (HASMCs) were cultured in calcification medium containing high phosphate (Pi) for 7 days in the absence or presence of an MMP-3 specific inhibitor. We found that adding MMP-3 inhibitor dose-dependently reduced Pi-induced calcium deposition as demonstrated by the O-cresolphthalein complexone assay. To further determine the importance of MMP-3 in arterial calcification, we utilized ex vivo aortic rings. In aortic rings from MMP-3 knockout mice, Pi-induced medial calcification was significantly reduced compared with wild-type mice. We next evaluated the effects of MMP-3 deletion in vivo . 12-week-old MMP-3 knockout and wild type mice were injected with 8mg/kg cholecalciferol and arteries were collected after 8 days, Calcium levels were significantly lower in MMP-3 knockout mice. Deletion of MMP-3 also reduced serum calcium levels, but not changed serum phosphate level. In concurrent studies, we showed by immunohistochemical staining that MMP-3 was highly expressed in calcified lesion from human tibial arteries. Conclusion: Together these findings suggest that MMP-3 promotes medial artery calcification, and that it may serve as a potential therapeutic target aimed at improving the poor outcomes of our patients with PAD.

The peptide hormone ghrelin is secreted in the stomach, with unique N-octanoylation at serine 3, which is a requirement for its functionality. These functions include growth hormone release, appetite stimulation, gastrointestinal motility, glucose regulation, and cell proliferation. The enzyme responsible for ghrelin acylation was recently identified as ghrelin O-acyltransferase (GOAT). In this study, porcine GOAT was cloned and characterized. A full-length cDNA of GOAT of 2013 bp was obtained, which included a 70-bp 5' UTR, a 635-bp 3' UTR, and a 1308-bp open reading frame encoding a protein of 415 amino acids. The GOAT and ghrelin mRNAs are co-expressed in stomach, pancreas, and duodenum at high levels. GOAT was also detected in liver, lung, brain, testis, spleen, kidney, heart, muscle, lipid, and ovary. Our results provide an important basis for further research on GOAT function and the relationship between ghrelin and GOAT.

Endogenous viruses integrate into the host genome and influence the expression of neighboring genes through their long terminal repeats (LTRs). In this study, we analyzed the promoter, enhancer and transcriptional activities of the chicken endogenous retrovirus ev21 LTR. The LTR was cloned into pGL3-basic and pGL3-promoter luciferase vectors in forward and reverse orientation separately. The luciferase activities were detected respectively in chicken embryonic fibroblast cell, human embryonic kidney cell line, human lung carcinoma cell line, Chinese hamster ovary cell line, and murine melanoma cell line. Relative luciferase activity analysis indicated that the ev21 LTR retained bi-directional promoter activity but no detectable enhancer activity in these cells. The constructs containing the LTR and F1 region show stronger promoter activity than the constructs containing only LTR. The transcriptional pattern of ev21 LTR varied in tissues of late feathering White Leghorn chicken at post-hatch day one. Skin exhibits the highest expression in the tissues examed. Collectively, our results indicate that the ev21 LTR exhibits tissue-type specific expression in White Leghorn chicken, and it also have regulatory potential.

PRNP is the gene encoding prion protein whose misfolded and β-sheet-rich isoform is the infectious agent of transmissible spongiform encephalopathy (TSE). TSE, also called prion diseases, cause fatal neurodegenerative and transmissible disorders in human and animals. Among these diseases, bovine spongiform encephalopathy (BSE) has tremendous impact on economy and human health in the world. In the present study, we hypothesize suppression of the PRNP gene expression could raise resistance to BSE in cattle by using vector-based small interfering RNA (siRNA) expression systems. Therefore, the objective was to screen effective DNA-encoding short hairpin RNAs (shRNAs) which could knockdown the PRNP gene expression in bovine fibroblast cells. Human U6 promoter was employed to drive shRNA transcription from the DNA vector, and seven shRNAs, that designed to target coding region and 3' untranslated region of the PRNP gene, were selected. Four out of seven shRNAs tested were found to be effective in inhibiting the PRNP gene expression, and the most significant suppression level was as much as 62.9% evidenced by real-time RT-PCR. Furthermore, the protein abundance was obviously reduced compared to the control. Overall, the present study demonstrated that vector-based siRNA expression systems is an efficient approach to knockdown the PRNP gene expression in bovine fibroblast cells and thereby provide donor cells for somatic cell nuclear cloning to produce cattle that is resistant to prion related diseases.

Background Medial artery calcification develops in diabetes, chronic kidney disease, and as part of the aging process. It is associated with increased morbidity and mortality in vascular patients. Bone morphogenetic proteins (BMPs) have previously been implicated in the initiation and progression of vascular calcification. We thus evaluated whether dorsomorphin homologue 1 (DMH1), a highly selective BMP inhibitor, could attenuate vascular calcification in vitro and in an organ culture model of medial calcification. Methods Confluent human aortic smooth muscle cells (SMCs) were cultured in calcification medium containing 3.0 mM inorganic phosphate (Pi) for 7 days with or without DMH1. Medial calcification was assessed using an aortic organ culture model. Calcification was visualized by alizarin red S staining, and calcium concentration was assessed by an o -cresolphthalein complexone calcium assay. Osteogenic cell and vascular SMC markers were determined by Western blot, quantitative reverse transcription polymerase chain reaction, and immunohistochemical staining. Results DMH1 reduced Pi-induced calcium deposition in human SMCs. It also antagonized human recombinant BMP2-induced calcium accumulation. Western blot further revealed that DMH1 was able to block Pi-mediated upregulation of the osteoblast markers osterix and alkaline phosphatase and downregulation of the SMC markers smooth muscle myosin heavy chain and SM22α as well as p-Smad1/5/8, suggesting that DMH1 may regulate SMC osteogenic differentiation through the BMP/Smad1/5/8 signaling pathway. Finally, using an ex vivo aortic ring organ culture model, we observed that DMH1 reduces Pi-induced aortic medial calcification. Conclusions The selective BMP inhibitor DMH1 can inhibit calcium accumulation in vascular SMCs and arterial segments exposed to elevated phosphate levels. Such small molecules may have clinical utility in reducing medial artery calcification in our population of vascular patients.

Objectives: Clinical studies have demonstrated associations between arterial calcification and adverse outcomes among patients with peripheral arterial disease (PAD). The causal relationship between calcification and the pathophysiology of ischemia, however, remains unclear. This study aims to evaluate the effects of vitamin D3-induced arterial calcification on functional recovery and resolution of ischemia in the rat hindlimb model. Methods: Arterial calcification was induced in 14 Sprague-Dawley rats using Vitamin D3. A control group of 14 rats received vehicle only. After 2 weeks to allow for calcification, unilateral hindlimb ischemia was induced by ligation and excision of the superficial femoral artery. Functional status and ischemia status were evaluated weekly for 1 month in a blinded manner. Results: 28 rats were assessed at all designated time periods. Arterial calcification was significantly greater in Vitamin D3-treated animals than in controls. Vitamin D3-treated rats had significantly worse total ischemia scores at each time point compared with controls. (Figure 1). All rats in the control group regained normal functional status and ischemia scores by week 4. Functional and ischemia scores in vitamin D3-treated animals did not return to baseline by the conclusion of the study. Conclusions: Vitamin D3 treatment significantly reduces functional recovery in a rat model of hindlimb ischemia. Future research aimed at understanding the relationship between vitamin D3 treatment, arterial calcification, and lower extremity ischemia may help to improve outcomes in patients with PAD.

Introduction Arterial calcification predicts accelerated restenosis after angioplasty and stenting. We sought to determine how calcification affected neointimal accumulation after balloon injury in the rat carotid. Hypothesis We propose that transformed SMCs in calcified arteries respond to balloon injury in an accelerated manner through a mechanism involving bone-related factors. Methods Arterial calcification was induced in male Sprague-Dawley rats weighing 350-400 grams by subcutaneous administration of vitamin D3. Control rats received vehicle. Both groups underwent left carotid balloon injury one week following injection, a time at which significant medial calcification has developed. Arteries were harvested twenty-one days following balloon injury. The degree of neointimal cell proliferation was next assessed by immunohistochemical staining for PCNA. Neointimal hyperplasia was measured by morphometric analysis using standard imaging analysis software. Results At 3 weeks after carotid balloon injury, mean cellular proliferation as measured by PCNA staining was higher in the intima of calcified than control arteries (63.8 ± 6.0 versus 40.5 ± 9.5 cells/hpf, P=ns). Calcified arteries from Vitamin D3-treated rats, however, had significantly more intimal hyperplasia than control uncalcified arteries (intima-to-media ratio = 1.374 ± 0.071 versus 0.758 ± 0.080, P=0.0021). Intimal and medial areas were also individually larger in calcified than control arteries. Conclusions Neointimal hyperplasia is accelerated in Vitamin D3-treated rats through a mechanism that partially involves increased SMC proliferation. Calcified vessels may respond differently to balloon angioplasty and this may explain the poorer results of endovascular interventions in patients with arterial calcification. Other factors including accelerated migration and increased matrix synthesis may also be involved. Treatments to inhibit restenosis in calcified arteries may differ from those that work in uncalcified vessels.

ATF4 is an osteoblast-enriched transcription factor of the leucine zipper family. We recently identified that vimentin, a leucine zipper-containing intermediate filament protein, suppresses ATF4-dependent osteocalcin (Ocn) transcription and osteoblast differentiation. Here we show that TGFβ inhibits ATF4-dependent activation of Ocn by up-regulation of vimentin expression. Osteoblasts lacking Atf4 (Atf4(-/-)) were less sensitive than wild-type (WT) cells to the inhibition by TGFβ on alkaline phosphatase activity, Ocn transcription and mineralization. Importantly, the anabolic effect of a monoclonal antibody neutralizing active TGFβ ligands on bone in WT mice was blunted in Atf4(-/-) mice. These data establish that ATF4 is required for TGFβ-related suppression of Ocn transcription and osteoblast differentiation in vitro and in vivo. Interestingly, TGFβ did not directly regulate the expression of ATF4; instead, it enhanced the expression of vimentin, a negative regulator of ATF4, at the post-transcriptional level. Accordingly, knockdown of endogenous vimentin in 2T3 osteoblasts abolished the inhibition of Ocn transcription by TGFβ, confirming an indirect mechanism by which TGFβ acts through vimentin to suppress ATF4-dependent Ocn activation. Furthermore, inhibition of PI3K/Akt/mTOR signaling, but not canonical Smad signaling, downstream of TGFβ, blocked TGFβ-induced synthesis of vimentin, and inhibited ATF4-dependent Ocn transcription in osteoblasts. Thus, our study identifies that TGFβ stimulates vimentin production via PI3K-Akt-mTOR signaling, which leads to suppression of ATF4-dependent Ocn transcription and osteoblast differentiation.