Expression and promoter analysis of a highly restricted integrin alpha gene in vascular smooth muscle

Abstract

Full genome annotation requires gene expression analysis and elucidation of promoter activity. Here, we analyzed the expression and promoter of a highly restricted integrin gene, Itga8. RNase protection and quantitative RT-PCR showed Itga8 to be expressed most abundantly in vascular smooth muscle cells (SMC). Transcription start site mapping of Itga8 revealed the immediate 5' promoter region to be poorly conserved with orthologous sequences in the human genome. Further comparative sequence analysis showed a number of conserved non-coding sequence modules around the Itga8 gene. The immediate promoter region and an upstream conserved sequence module were each found to contain a CArG box, which is a binding site for serum response factor (SRF). Luciferase reporter assays revealed activity of several Itga8 promoter constructs with no apparent restricted activity to SMC types. Further, neither SRF nor its coactivator, Myocardin (MYOCD), was able to induce several distinct Itga8 promoter constructs. Transgenic mouse studies failed to reveal Itga8 promoter activity indicating distal regulatory elements likely control this gene's in vivo expression profile. Interestingly, although the promoter was unresponsive to SRF/MYOCD, the endogenous Itga8 gene showed increases in expression upon ectopic MYOCD expression even though knockdown of SRF both in vitro and in vivo failed to demonstrate a corresponding change in Itga8. Collectively, these data demonstrate that Itga8 expression is CArGSRF independent, but MYOCD dependent through an as yet unknown sequence module that is distal from the promoter region.

Cadherin-11 is a novel regulator of extracellular matrix synthesis and tissue mechanics

“…MRTF-A has been shown to control Col1A2 promoter activity as well as expression of other extracellular matrix molecules (Asparuhova et al, 2011;Luchsinger et al, 2011). MYOCD has also been implicated in studies showing CArG-RE-independent regulation of ECM (Tang et al, 2008;Kitchen et al, 2013). Interestingly, engagement of CDH11 on surface-immobilized CDH11-Fc increased the mRNA levels of all these transcription factors significantly.…”

“…In agreement, knocking down either MYOCD or MRTF-A and -B decreased CDH11-mediated expression of collagen and elastin, but the effects of MRTF-A and -B were significantly stronger. Although past studies have only recognized MRTF and MYOCD as co-activators of SRF, some recent studies have shown that they can act as transcription factors independently (Tang et al, 2008;Asparuhova et al, 2011;Luchsinger et al, 2011;Smith et al, 2012;Kitchen et al, 2013). MRTF-A has been shown to control Col1A2 promoter activity as well as expression of other extracellular matrix molecules (Asparuhova et al, 2011;Luchsinger et al, 2011).…”

Ca2+/calmodulin‐dependent protein kinase II‐γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling

“…The strong correlation observed in the present study between CaMKIIγ expression in dedifferentiating VSM after injury and differentiating VSM in vitro in response to myocardin overexpression provides some clues in the case of CaMKIIγ. More studies are needed to determine whether myocardin directly regulates CaMKIIγ through an SRF‐independent mechanism, such as is the case for a8‐integrin (45), or indirectly regulates expression through other mechanisms, such as mRNA stability, epigenetic modifications, micro‐RNA, or long noncoding RNA.…”

The short and long of noncoding sequences in the control of vascular cell phenotypes

“…Thus, the CArG-SRF-MYOCD triad is the primary transcriptional switch directing the SMC differentiated state. Of note, not all genes expressed in differentiated SMCs contain functional CArG boxes (bold genes in Table 1), and MYOCD is not sufficient to activate some of these [53, 54], although at least one SMC-restricted gene ( ITGA8 ) appears to be activated by MYOCD in a CArG-SRF-independent manner [55]. Understanding how MYOCD performs such SRF-independent actions of transcription is an open area for future investigation.…”