EGLN3 prolyl hydroxylase regulates skeletal muscle differentiation and myogenin protein stability

EGLN3, a member of the EGLN family of prolyl hydroxylases, has been shown to catalyze hydroxylation of the alpha subunit of hypoxia-inducible factor-alpha, which targets hypoxia-inducible factor-alpha for ubiquitination by a ubiquitin ligase complex containing the von Hippel-Lindau (VHL) tumor suppressor. We now report that EGLN3 levels increase during C2C12 skeletal myoblast differentiation. EGLN3 small interference RNAs and EGLN3 antisense oligonucleotides blocked C2C12 differentiation and decreased levels of myogenin, a member of the MyoD family of myogenic regulatory factors, which plays a critical role in myogenic differentiation. We also report that EGLN3 interacts with and stabilizes myogenin protein, whereas VHL associates with and destabilizes myogenin via the ubiquitin-proteasome system. The effect of VHL on myogenin stability and ubiquitination can be reversed, at least in part, by overexpression of EGLN3, suggesting that its binding to myogenin may prevent VHL-mediated degradation. These data demonstrate a novel role for EGLN3 in regulating skeletal muscle differentiation and gene expression. In addition, this report provides evidence for a novel pathway that regulates myogenin expression and skeletal muscle differentiation.     

Serum response factor, its cofactors, and epithelial-mesenchymal signaling in urinary bladder smooth muscle formation

Little is known about the mechanism of bladder smooth muscle differentiation. We hypothesize that epithelial-mesenchymal signaling induces the expression of smooth muscle proteins in bladder mesenchyme resulting in smooth muscle differentiation. We confirmed that smooth muscle differentiation in the mouse urinary bladder occurs first at gestational day 14 (E14) based upon immunohistochemical localization of smooth muscle alpha-actin (SMAA). To investigate murine bladder smooth muscle differentiation and epithlelial-mesenchymal signaling in the developing bladder, we analyzed gene expression profiles of intact embryonic murine bladders and separated epithelial and mesenchymal components at embryonic days E13, E14, E15, E16, and postnatal day 1 (P1). Using cDNA microarray, we identified regulators of vascular smooth muscle differentiation in bladder mesenchyme, including serum response factor (SRF) and its cofactors, ELK1 and SRF accessory protein (SAP)1, as well as two SRF-associated pathways, angiotension receptor II and transforming growth factor- beta2. Immunohistochemistry showed diffuse expression of SRF in the bladder at E12 with localization of expression to the peripheral mesenchyme at E13 and E14. Our results suggest that bladder smooth muscle differentiation may share a similar gene expression program as occurs during vascular smooth muscle differentiation. The unique structure of the urinary bladder makes it an ideal model for studies of smooth muscle differentiation and epithelial-mesenchymal signaling.     

Striated muscle preferentially expressed genes alpha and beta are two serine/threonine protein kinases derived from the same gene as the aortic preferentially expressed gene-1

Aortic preferentially expressed gene (APEG)-1 is a 1.4-kilobase pair (kb) mRNA expressed in vascular smooth muscle cells and is down-regulated by vascular injury. An APEG-1 5'-end cDNA probe identified three additional isoforms. The 9-kb striated preferentially expressed gene (SPEG)alpha and the 11-kb SPEGbeta were found in skeletal muscle and heart. The 4-kb brain preferentially expressed gene was detected in the brain and aorta. We report here cloning of the 11-kb SPEGbeta cDNA. SPEGbeta encodes a 355-kDa protein that contains two serine/threonine kinase domains and is homologous to proteins of the myosin light chain kinase family. At least one kinase domain is active and capable of autophosphorylation. In the genome, all four isoforms share the middle three of the five exons of APEG-1, and they differ from each other by using different 5'- and 3'-ends and alternative splicing. We show that the expression of SPEGalpha and SPEGbeta is developmentally regulated in the striated muscle during C2C12 myoblast to myotube differentiation in vitro and cardiomyocyte maturation in vivo. This developmental regulation suggests that both SPEGalpha and SPEGbeta can serve as sensitive markers for striated muscle differentiation and that they may be important for adult striated muscle function.     

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.     

ESDN, a novel neuropilin-like membrane protein cloned from vascular cells with the longest secretory signal sequence among eukaryotes, is up-regulated after vascular injury.

A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins.     

Differential expression and developmental regulation of a novel alpha-dystrobrevin isoform in muscle

Alpha-dystrobrevin is a dystrophin-related protein expressed primarily in skeletal muscle, heart, lung and brain. In skeletal muscle, alpha-dystrobrevin is a component of the dystrophin-associated glycoprotein complex and is localized to the sarcolemma, presumably through interactions with dystrophin and utrophin. Alternative splicing of the alpha-dystrobrevin gene generates multiple isoforms which have been grouped into three major classes: alpha-DB1, alpha-DB2, and alpha-DB3. Various isoforms have been shown to interact with a variety of proteins; however, the physiological function of the alpha-dystrobrevins remains unknown. In the present study, we have cloned a novel alpha-dystrobrevin cDNA encoding a protein (referred to as alpha-DB2b) with a unique 11 amino acid C-terminal tail. Using RT PCR with primers specific to the new isoform, we have characterized its expression in skeletal muscle, heart, and brain, and in differentiating C2C12 muscle cells. We show that alpha-DB2b is expressed in skeletal muscle, heart and brain, and that exons 12 and 13 are alternatively spliced in alpha-DB2b to generate at least three splice variants. The major alpha-DB2b splice variant expressed in adult skeletal muscle and heart contains exons 12 and 13, while in adult brain, two alpha-DB2b splice variants are expressed at similar levels. This is consistent with the preferential expression of exons 12 and 13 in other alpha-dystrobrevin isoforms in skeletal muscle and heart. Similarly, in alpha-DB1 the first 21 nucleotides of exon 18 are preferentially expressed in skeletal muscle and heart relative to brain. We also show that the expression of alternatively spliced alpha-DB2b is developmentally regulated in muscle; during differentiation of C2C12 cells, alpha-DB2b expression switches from an isoform lacking exons 12 and 13 to one containing them. We demonstrate similar developmental upregulation of exons 12, 13, and 18 in alpha-DB1 and of exons 12 and 13 in alpha-DB2a. Finally, we show that alpha-DB2b protein is expressed in adult skeletal muscle, suggesting that it has a functional role in adult muscle. Together, these data suggest that alternatively spliced variants of the new alpha-dystrobrevin isoform, alpha-DB2b, are differentially expressed in various tissues and developmentally regulated during muscle cell differentiation in a fashion similar to that previously described for alpha-dystrobrevin isoforms.     

Prolyl Hydroxylase EGLN3 Regulates Skeletal Myoblast Differentiation through an NF-��B-dependent Pathway

The egg-laying abnormal-9 (EGLN) prolyl hydroxylases have been shown to regulate the stability and thereby the activity of the α subunits of hypoxia-inducible factor (HIF) through its ability to catalyze their hydroxylation. We have previously shown that EGLN3 promotes differentiation of C2C12 skeletal myoblasts. However, the mechanism underlying this effect remains to be fully elucidated. Here, we report that exposure of C2C12 cells to dimethyl oxalylglycine (DMOG), desferrioxamine, and hypoxia, all inhibitors of prolyl hydroxylase activity, led to repression of C2C12 myogenic differentiation. Inactivation of HIF by expression of a HIF dominant-negative mutant or deletion of HIF-1α by RNA interference did not affect the inhibitory effect of DMOG, suggesting that the effect of DMOG is HIF-independent. Pharmacologic inactivation of EGLN3 hydroxylase resulted in activation of the canonical NF-κB pathway. The inhibitory effect of DMOG on myogenic differentiation was markedly impaired in C2C12 cells expressing a dominant-negative mutant of IκBα. Exogenous expression of wild-type EGLN3, but not its catalytically inactive mutant, significantly inhibited NF-κB activation induced by overexpressed TRAF2 or IκB kinase 2. In contrast, deletion of EGLN3 by small interfering RNAs led to activation of NF-κB. These data suggest that EGLN3 is a negative regulator of NF-κB, and its prolyl hydroxylase activity is required for this effect. Furthermore, wild-type EGLN3, but not its catalytically inactive mutant, potentiated myogenic differentiation. This study demonstrates a novel role for EGLN3 in the regulation of NF-κB and suggests that it is involved in mediating myogenic differentiation, which is HIF-independent.     

Notch2 and Notch3 function together to regulate vascular smooth muscle development

Notch signaling has been implicated in the regulation of smooth muscle differentiation, but the precise role of Notch receptors is ill defined. Although Notch3 receptor expression is high in smooth muscle, Notch3 mutant mice are viable and display only mild defects in vascular patterning and smooth muscle differentiation. Notch2 is also expressed in smooth muscle and Notch2 mutant mice show cardiovascular abnormalities indicative of smooth muscle defects. Together, these findings infer that Notch2 and Notch3 act together to govern vascular development and smooth muscle differentiation. To address this hypothesis, we characterized the phenotype of mice with a combined deficiency in Notch2 and Notch3. Our results show that when Notch2 and Notch3 genes are simultaneously disrupted, mice die in utero at mid-gestation due to severe vascular abnormalities. Assembly of the vascular network occurs normally as assessed by Pecam1 expression, however smooth muscle cells surrounding the vessels are grossly deficient leading to vascular collapse. In vitro analysis show that both Notch2 and Notch3 robustly activate smooth muscle differentiation genes, and Notch3, but not Notch2 is a target of Notch signaling. These data highlight the combined actions of the Notch receptors in the regulation of vascular development, and suggest that while these receptors exhibit compensatory roles in smooth muscle, their functions are not entirely overlapping.     

A novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in rat vascular smooth muscle and renal proximal tubular cells

Angiotensin II stimulates cellular hypertrophy in cultured vascular smooth muscle and renal proximal tubular cells. This effect is believed to be one of earliest morphological changes of heart and renal failure. However, the precise molecular mechanism involved in angiotensin II-induced hypertrophy is poorly understood. In the present study we report the isolation of a novel angiotensin II type 1 receptor-associated protein. It encodes a 531-amino acid protein. Its mRNA is detected in all human tissues examined but highly expressed in the human kidney, pancreas, heart, and human embryonic kidney cells as well as rat vascular smooth muscle and renal proximal tubular cells. Protein synthesis and relative cell size analyzed by flow cytometry studies indicate that overexpression of the novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in cultured rat vascular smooth muscle and renal proximal tubular cells. In contrast, the hypertrophic effects was reversed in renal proximal tubular cell lines expressing the novel gene in the antisense orientation and its dominant negative mutant, which lacks the last 101 amino acids in its carboxyl-terminal tail. The hypertrophic effects are at least in part mediated via protein kinase B activation or cyclin-dependent kinase inhibitor, p27(kip1) protein expression level in vascular smooth muscle, and renal proximal tubular cells. Moreover, angiotensin II could not stimulate cellular hypertrophy in renal proximal tubular cells expressing the novel gene in the antisense orientation and its mutant. These findings may provide new molecular mechanisms to understand hypertrophic agents such as angiotensin II-induced cellular hypertrophy.     

microRNAs参与韧带成纤维细胞成骨分化相关分子表达的调节

韧带成纤维细胞成骨分化与强直性脊柱炎等多种异位骨化相关性疾病有关,但其机制尚不清楚.本研究目的在于观察韧带成纤维细胞在成骨分化过程中microRNA和mRNA的表达谱变化,以期为揭示成纤维细胞成骨分化机制提供研究基础.原代培养韧带成纤维细胞、地塞米松、抗坏血酸和β 磷酸甘油体外诱导其成骨分化, RT-PCR检测成骨标志物骨钙素和Runx2的表达.采用表达谱基因芯片分析诱导0、7、14 d后韧带成纤维细胞的microRNAs和mRNAs表达,并用实时定量PCR（RT-qPCR）和Western印迹方法验证生物信息学分析结果.结果显示,与未诱导前相比,成骨分化第7 d有66个microRNAs和640个mRNA表达上调,94个microRNAs和744个mRNA表达下调;成骨分化第14 d有58个microRNAs和781个mRNA表达上调,96个microRNAs和603个mRNA表达下调.实时定量PCR和Western印迹验证结果显示,miR-29b在成骨分化过程中表达上调,TGFβ3表达水平降低,与芯片结果一致,miR-29b通过抑制TGFβ3蛋白的翻译来促进Runx2的表达,从而促进韧带成纤维细胞向成骨细胞分化.韧带成纤维细胞成骨分化过程中,microRNA调控基因及mRNA差异表达基因除涉及BMPs、Wnt和Ihh等信号通路外,在成纤维细胞成骨分化过程中可能还存在其它的新机制.     

Peroxisome proliferator-activated receptor delta is up-regulated during vascular lesion formation and promotes post-confluent cell proliferation in vascular smooth muscle cells.

Although peroxisome proliferator-activated receptor (PPAR) delta is widely expressed in many tissues, the role of PPARdelta is poorly understood. In this study, we report that PPARdelta was up-regulated in vascular smooth muscle cells (VSMC) during vascular lesion formation. By using Northern blot analysis, we demonstrated that PPARdelta was increased by 3-4-fold in VSMC treated with platelet-derived growth factor (PDGF) (20 ng/ml). In addition, PDGF-induced PPARdelta mRNA expression neither needs de novo protein synthesis nor affects the stability of PPARdelta mRNA in VSMC. Preincubation of VSMC with phosphatidylinositol 3-kinase inhibitor (LY294002, 50 micromol/liter) or infection of VSMC with an adenovirus carrying the gene for a dominant negative form of Akt abrogated PDGF-induced PPARdelta mRNA expression, suggesting that phosphatidylinositol 3-kinase/Akt signaling pathway is involved in the regulation of PDGF-induced PPARdelta mRNA expression in VSMC. To explore the role of PPARdelta in VSMC, we generated rat vascular smooth muscle cells (A7r5) stably overexpressing PPARdelta and the control green fluorescent protein. Overexpression of PPARdelta in VSMC increased post-confluent cell proliferation by increasing the cyclin A and CDK2 as well as decreasing p57(kip2). Taken together, the results suggest that PPARdelta plays an important role in the pathology of diseases associated with VSMC proliferation, such as primary atherosclerosis and restenosis.     

Characterization of smooth muscle differentiation of purified human skeletal muscle‐derived cells

The purpose of this study is to characterize the smooth muscle differentiation of purified human muscle‐derived cells (hMDCs). The isolation and purification of hMDCs were conducted by modified preplate technique and Dynal CD34 cell selection. Smooth muscle cell differentiation was induced by the use of smooth muscle induction medium (SMIM) and low‐serum medium. The gene expressions at the mRNA and protein levels of undifferentiated and differentiated hMDCs were tested by RT‐PCR, Western blot and immunofluorescence studies. Western blot and immunofluorescence studies demonstrated the purified hMDCs cultured in SMIM for 4 weeks and expressed significant amount of smooth muscle myosin heavy chain (MHC) and α‐smooth muscle actin (ASMA). The cells cultured in low‐serum medium for 4 weeks also expressed ASMA, while the control group did not. RT‐PCR analysis showed increased gene expression of smooth muscle markers, such as ASMA, Calponin, SM22, Caldesmon, Smoothelin and MHC when purified hMDCs were exposed to SMIM for 2 and 4 weeks when compared to the controls. In conclusion, we confirmed the smooth muscle differentiation capability of purified hMDCs. The gene expression of smooth muscle differentiation of purified hMDCs was characterized. These cells may be potential biomaterials for human tissue regeneration.