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To investigate the role of cell-cell interactions between keratinocytes and fibroblasts at the dermal-epidermal junction in the regulation of extracellular matrix protein expression, we have developed a cell coculture model that allows both cell types to interact on a reciprocal basis and yet be isolated as pure populations for quantitative analysis. Using porous membrane inserts as a substrate for keratinocytes grown in coculture over fibroblast monolayers, we report that coculture stimulates cell growth and total protein synthesis in both cell types when compared to monocultured controls. Both keratinocytes and fibroblasts synthesize laminin B1 and B2 chains with an additional subunit comigrating with laminin M chain detected in keratinocytes but only faintly visible in fibroblasts. Laminin A chain synthesis could not be detected. Although laminin subunit composition did not change in either cell type, fractional laminin synthesis increases by 41.0 +/- 2.3% in cocultured keratinocytes and decreases by 73.8 +/- 8.1% in cocultured fibroblasts when compared to monocultured controls. In cocultured keratinocytes, steady-state mRNA levels for laminin B1, B2, and M chains increased by 69.4, 63.5, and 136.8%, respectively, when compared to monocultured controls. However, in cocultured fibroblasts, laminin B1 and B2 chain mRNA decreased by 74.2 and 72.7%, respectively. Laminin M chain mRNA could not be detected in fibroblasts. These results suggest that synthesis and expression of laminin is regulated through reciprocal cell-cell interactions in fibroblasts and keratinocytes grown in coculture.

Our objectives were to 1) determine how growth state and cell density affect the expression of the smooth muscle (SM) and nonmuscle (NM) isoforms of the 20-kDa myosin regulatory light chains (MLC20) in cultured rat aortic smooth muscle cells (SMC) and 2) to determine whether angiotensin II stimulates differential phosphorylation of SM and NM MLC20 isoforms in an effort to assess whether the SM and NM isoforms may subserve different cellular functions. The results demonstrated that changes in the SM MLC20 isoform content were inversely correlated with cell growth but independent of cell density. MLC20 phosphorylation levels were 20.8 +/- 2.9 and 19.4 +/- 3.7% for SM and NM isoforms, respectively, in unstimulated, substrate-attached SMC. Angiotensin II transiently elevated phosphorylation levels of both the SM and NM MLC20 isoforms to 60-70%. No differences in either the magnitude or the kinetics of phosphorylation were observed for the SM vs. NM isoforms. Forskolin, 3-isobutyl-1-methylxanthine, or isoproterenol treatment led to parallel dephosphorylation of the SM- and NM-specific isoforms followed by depolymerization of stress fibers and cell arborization. The studies provide evidence that growth arrest of cultured SMC enhances expression of cell-specific/-selective proteins characteristic of differentiated SM. However, there was no evidence for differential phosphorylation changes of SM and NM MLC20 isoforms in response to activating or relaxing agents as expected if these isoforms subserve different cellular functions.

We have compiled evidence that nonmuscle isoforms of both myosin heavy chain (NM MHC) and myosin regulatory light chain (NM LC20) are present in fully differentiated smooth muscles (SM). In swine carotid media sodium dodecyl sulfate-gel electrophoresis separated three MHC bands. The upper two bands were identified by immunoblotting as SM-specific isoforms. The lowest MHC band amounted to 14 +/- 2% of the total MHC and was electrophoretically and antigenically similar to platelet MHC. Two-dimensional gel electrophoresis of swine carotid media extracts resolved multiple LC20 species, including phosphorylated and "satellite" forms. Mass spectrometric analysis of tryptic peptides from blots of these gels demonstrated two LC20 isoforms. The measured peptide masses correspond with two published cDNA sequences proposed to represent SM and NM LC20 isoforms. These sequences readily explain the electrophoretic behavior of the isoforms. The minor isoform's abundance (16 +/- 3%, corresponding to NM MHC), antigenic properties, and pattern of expression in tissue culture all confirm that this is a NM LC20 isoform. The localization and functional significance of NM myosin in smooth muscle is unknown.