Abstract
Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.
Highlights
Smooth muscle cells (SMCs)3 are the major contractile component of the vascular, respiratory, genitourinary, and digestive systems
For testing the effects of smooth muscle gene expression by depletion of endogenous UBR5, control siRNA or UBR5 siRNA were transfected into A10 cells for 48 h and total RNA was harvested for quantitative real timePCR or protein was extracted for Western blotting as described below
UBR5 Localizes to the Nucleus of Smooth Muscle Cells and Interacts with Myocardin in Vivo and in Vitro—As myocardin is a potent transcription factor that can convert most cells to SMC-like cells (26), we hypothesize that myocardin function is likely to be regulated through its association with other proteins in these non-SMCs
Summary
Yeast Two-hybrid Screen—A fragment of the mouse myocardin cDNA encoding the NH2-terminal 585 amino acids was cloned into the bait vector pAS2-1 (Promega). A variety of UBR5 fragments, as described, were amplified by PCR and fused to the SV40 nuclear location sequence LYPKKKRKGVEDQYK at the 3Ј terminus of the coding sequence to force expression in cell nucleus Those fragments were ligated to pcDNA3.1His (Invitrogen), resulting in expression of a fusion protein with amino-terminal His and Omni epitope tags. CDNA encoding the COOH-terminal end of the UBR5 HECT domain (2337–2799 amino acids) was cloned into pET28 vectors (Novagen) to generate T7 fusion proteins These GST or T7 fusion proteins were produced in Escherichia coli BL21-star (Stratagene) cells and GST pulldown assays were carried out as reported in our previous study (11). For testing the effects of smooth muscle gene expression by depletion of endogenous UBR5, control siRNA or UBR5 siRNA were transfected into A10 cells for 48 h and total RNA was harvested for quantitative real time (qRT)PCR or protein was extracted for Western blotting as described below. The right and left carotid arteries were harvested 5 and 10 days after injury and total RNA was extracted with RNAqueous-Micro kit (Ambion). cDNA was reverse transcribed and gene expression was measured by qRTPCR as described above
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