Density‐Dependent modulatioon of vascular smooth muscle α‐actin biosynthetic processing in differntiated BC3H1 myogenic cells

Abstract

The expression of vascular smooth muscle (VSM) alpha-actin mRNA during BC3H1 myogenic cell differentiation is specifically stimulated by conditions of high cell density. Non-proteolytic dissociation of cell-cell and cell-matrix contacts in post-confluent cultures of BC3H1 myocytes using EDTA promotes loss of the differentiated morphological phenotype. EDTA-dispersed myocytes exhibit an undifferentiated fibroblastoid appearance and contained reduced levels of both VSM and skeletal alpha-actin mRNA. Muscle alpha-actin mRNA levels in EDTA-dispersed myocytes were not restored to that observed in confluent myocyte preparations by experimental manipulation of cell density conditions. Pulse-labeling techniques using L-[35S]cysteine to identify muscle actin biosynthetic intermediates revealed that EDTA-dispersed myocytes expressed nascent forms of both the VSM and skeletal muscle alpha-actin polypeptide chains. However EDTA-dispersed myocytes were less efficient in the post-translational processing of immature VSM alpha-actin compared to non-dispersed myocytes. Simple cell-to-cell contact may mediate VSM alpha-actin processing efficiency since high-density preparations of EDTA-dispersed myocytes processed more VSM alpha-actin intermediate than myocytes plated at low density. The actin isoform selectivity of the response to modulation of intercellular contacts suggests that actin biosynthesis in BC3H1 myogenic cells involves mechanisms capable of discriminating between different isoform classes of nascent actin polypeptide chains.

Proteoglycan biosynthesis is required in BC3H1 myogenic cells for modulation of vascular smooth muscle α‐actin gene expression in response to microenvironmental signals

“…Delaying the exposure of quiescent cells to BDX by 24 hours (3-day time points) or 48 hours (4-day time points) after the addition of N2 medium was less inhibitory and produced a 7-fold and 4.6-fold reduction, respectively, in the amount of a-actin mRNA detected relative to control preparations treated with DMSO alone. Since myocytes maintained a full 4 days after the switch to N2 differentiation medium begin to express a small amount of skeletal a-actin mRNA that exhibits a size similarity to VSM a-actin (Strauch and Reeser, 19891, we also performed Northern blot analysis using a 3'-UT region cDNA probe that specifically hybridizes to mouse VSM a-actin mRNA (Min et al, 1988;Strauch et al, 1992) to determine if the BDX-resistant a-actin mRNA detected in fully differentiated myocytes represented novel actin transcripts that were unrelated to VSM a-actin mRNA which is the only a-actin expressed in earlier stage cells. We and others (Edmondson and Olson, 1989) have not observed cardiac a-actin expression in BC3H1 myogenic cells using standard culture conditions.…”

“…Nuclear run-on studies of actin mRNA transcription was performed essentially as described in detail by Ausubel et al (1993) and Strauch et al (1992). BC3H1 cells cultured in the presence or absence of p-D-xyloside were extracted in lysis buffer containing 10 mM Tris-HCI, pH 7.4, 10 mM NaC1, 3 mM MgC12, and 0.5% NP-40, and centrifuged at 500 x g for 5 minutes a t 4°C to sediment cell nuclei.…”