Abstract
Magicin, a protein that we isolated earlier as an interactor of the neurofibromatosis 2 protein merlin, was independently identified as MED28, a subunit of the mammalian Mediator complex. Mediator complex is an evolutionarily conserved transcriptional cofactor, which plays an essential role in positive and negative gene regulation. Distinct Mediator subunit composition is thought to contribute to gene regulation specificity based on the interaction of specific subunits with subsets of transcription factors. Here we report that down-regulation of Med28 expression in NIH3T3 cells results in a significant induction of several genes associated with smooth muscle cell (SMC) differentiation. Conversely, overexpression of MED28 represses expression of SMC genes, in concordance with our knockdown data. More importantly, multipotent mesenchymal-derived murine precursors can transdifferentiate into SMCs when Med28 is down-regulated. Our data also show that Med28 functions as a negative regulator of SMC differentiation in concert with other Mediator subunits including Med6, Med8, and Med18 within the Mediator head module. Our results provide strong evidence that MED28 may function as a scaffolding protein by maintaining the stability of a submodule within the head module and that components of this submodule act together in a gene regulatory program to suppress SMC differentiation. The results presented here demonstrate for the first time that the mammalian Mediator subunit MED28 functions as a repressor of SMC differentiation, which could have implications for disorders associated with abnormalities in SMC growth and differentiation, including atherosclerosis, asthma, hypertension, and smooth muscle tumors.
Highlights
We previously identified a novel protein, which we named magicin, as a specific binding partner for the cytoskeletal neurofibromatosis 2 tumor suppressor protein merlin
MED28 as a novel merlin-interacting protein, we showed an up-regulation by RT-PCR, further confirming observed Med28 in association with the actin cytoskeleton of that the expression of Med28 negatively regulates SM-specific the mouse neuronal (Cath. a-differentiated) cell line as deter- genes (Fig. 2B)
MED28, which we originally identified as a merlin interactor, is established as one of the head module subunits [16, 17]
Summary
Reagents and Antibodies—Human full-length MED28 (hMED28) was cloned into the pEGFP-N1 (Clontech) plasmid, as well as the CSCW2 lentiviral plasmid. Primary antibodies used included the anti-MED28 antibodies Tim (rabbit polyclonal) and 7E1 (mouse monoclonal), which have been previously described [1]; M2 (anti-FLAG monoclonal; Sigma), MED6, CDK8, and SM22␣/transgelin (Santa Cruz Biotechnology); SM-␥Actin (MP Biomedicals); GAPDH (Chemicon); and SM-␣Actin, ␣-tubulin, and normal rabbit serum (Sigma). Real-time RT-PCR was carried out to analyze Med expression, as well as the SM differentiation cofactor cysteine-rich protein 1 (CRP1) (Csrp1) and SM differentiation markers including SM22␣ (Tagln), SM-␣Actin (Acta2), and SM-␥Actin (Actg). To perform realtime RT-PCR, first-strand cDNA was combined with iQ SYBR Green supermix (Bio-Rad) and mouse sequence-specific primers for Med, CRP1, SM22␣, SM-␣Actin, and SM-␥Actin, as well as the control housekeeping gene GAPDH. Standard RT-PCR using mouse-specific primers was carried out for 27–35 cycles to visualize changes in expression for Med, SM22␣, SM-␥Actin, SM-␣Actin, CRP1, and GAPDH, as well as the definitive smooth muscle marker SM-.
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