Cultured aortic smooth muscle cells (SMC) exhibit morphological and phenotypic modulation characterized by a change from a substrate attached monolayer culture to a multilayered nodular cell culture. in which SMC are embedded into the extracellular matrix (ECM). Associated with nodule formation is a change in the pattern of SMC gene expression including increased expression of a well-characterized marker of SMC differentiation, SM-actin, and a Mr=38,000Da glycoprotein (gp38k). gp38k has sequence homology with proteins reported to be correlated with tissue remodeling. To characterize the gp38k mRNA we designed degenerate oligonucleotides based on partial polypeptide sequences and selected a cDNA encoding the full-length gp38k. Southern analysis indicates that porcine gp38k is present as a signal copy gene. Northern and western analyses indicate that an increase in gp38k expression is correlated with an increase in the steady state level of gp38k mRNA; and is present in cultures that have initiated the formation of multilayered foci and nodules. The correlation between SMC differentiation and gp38k expression is further established by using culture conditions that facilitate SMC differentiation. Cultures seeded onto reconstituted ECM show rapid formation of nodules and increased expression of gp38k mRNA. Comparison of the gp38k cDNA sequences with nucleotide and protein sequences available through GenBank reveals that nolecules homologous to gp38k were present in human, mouse, bovine, and Drosophila tissues suggesting that gp38k may be a member of a gene family. Although a function for gp38k has not been identified, its expression is correlated with a specific process important in phenotypic and morphological modulation of vascular SMC. NIH-HL40417.

CLUSTERIN EXPRESSION AND NODULE FORMATION IN CLONED PORCINE VASCULAR SMOOTH MUSCLE CELLS.

Clusterin is a heterodimeric glycoprotein, expressed by various mammalian cell types and shown to have cell-cell adhesion promoting activity. In culture, porcine vascular smooth muscle cells (SMC) undergo morphological and phenotypic modulation associated with a change from a substrate attached monolayer culture to a nodular culture in which most of the cells are present in multicellular aggregations (nodules). Our previous studies demonstrated that nodular SMC cultures express relatively high levels of clusterin mRNA and protein [Thomas-Salgar and Millis (1994) JBC 269:17879]. To evaluate the possibility that clusterin is required for nodule formation porcine SMC were co-transfected with pEMSVscribe2 containing a 1.7kb full length murine clusterin cDNA insert in antisense orientation and pRSVneo using calcium phosphate coprecipitation and selected with 500g/ml G418. G418 resistant clones were subsequently cloned and cultured. G418 resistant clones were seeded at equal density, cultured for 9 days, and screened for clusterin production by western immunoassay. Clones expressing a reduced level of endogenous clusterin (asCLU13) or high level of endogenous clusterin (asCLU18) were identified and compared with uncloned SMC in a nodule forming assay on Matrigel (contains clusterin) or collagen-gel (lacks clusterin). Even after 50hr of incubation clone asCLU13 did not form nodular aggregations on collagen-gel, but formed nodules within 9-20hr on clusterin-containing Matrigel. Clone asCLU18 formed nodules within 20hr on collagen-gel and within 9-20 hr on Matrigel. These results demonstrate that clusterin is a critical factor in SMC nodule formation. Supported by NYS American Heart Association.

CLUSTERIN REGULATES PHENOTYPIC MODULATION (NODULE FORMATION) IN VASCULAR SMOOTH MUSCLE CELLS.

A.J.T. Millis and C.L. Moulson, and Center for the Study of Comparative Functional Genomics,, Biology Department, University at Albany, SUNY - Albany, Albany, NY 12222.

EXPRESSION OF METALLOPROTEINASE GENES DURING CELLULAR AGING IS REGULATED BY ENDOGENOUS TGFb ACTIVITY

G. Zeng, H. M. McCue and A. Millis Center for Cellular Differentiation, Biology Department, University at Albany, SUNY - Albany, Albany, NY 12222.

In culture, non-transformed human diploid fibroblasts divide a limited number of times resulting in a non-proliferating senescent cell culture which exhibits an altered pattern of gene expression. An event preceeding the onset of sensecence is an increase in the expression of mRNAs for two metalloproteinases; collagenase (CL) and stromelysin (SL). In contrast, the expression of the mRNA for an inhibitor of metalloproteinases, TIMP-1, is diminished [Millis, et al., 1992. ECR 201:373]. Because the cytokine TGF1 is known to regulate the expression of each of those three genes and to be synthesized and secreted by human fibroblasts we tested the hypothesis that the age-specific modulation of CL, SL, and TIMP-1 were regulated by endogenous TGF by using an activity-neutralizing antibody. In young cell cultures exposure to the TGF neutralizing antibody resulted in a significant increase in CL and SL expression and decrease in TIMP-1expression as eveidenced by western immunoblot assay. The antibody did not affect gene expression in pre-senescent cell cultures although those cultures retained the abiltiy to respond to added TGF1 via modulation of CL, SL, and TIMP-1 expression. Quantification of the levels of active TGF, using a growth inhibition assay and an ELISA, indicates that the level of active TGF1 is decreased during replicative senescence supporting the conclusion that modulation of CL, SL, and TIMP-1 expression results from modification of endogenous TGF activity. [Supported by AG09279 from the NIH-NIA]