Expression of porcine complement cytolysis inhibitor mRNA in cultured aortic smooth muscle cells. Changes during differentiation in vitro.

Porcine smooth muscle cells (SMC) grown to a high density monolayer culture undergo a morphological transition in which the cells draw away from the substrate and form multicellular nodules. The cells within the nodule resemble SMC in the aortic media and in some atherosclerotic plaques. The process of nodule formation is associated with the enhanced production of a secreted 38-kDa glycoprotein. To characterize the 38-kDa protein and its expression, a cDNA clone (pc38K) was isolated by immunological screening of an expression library. The 1646-base pair cDNA contains a single open reading frame encoding 446 amino acids. This sequence shows 72% homology with the human complement cytolysis inhibitor (CLI), also called serum protein-40,40, and 68% identity with rat sulfated glycoprotein-2. Based on this homology, we refer to the protein encoded by pc38K as CLI. This polypeptide includes a potential signal sequence, seven glycosylation sites and 10 cysteines in two clusters of five each. Southern blot analysis reveals that a single copy gene encoding CLI is present in mammals and chicken. In Northern blot analysis of SMC RNA, pc38K hybridizes to a mRNA of about 1.9 kilobases that is preferentially expressed in nodular SMC. The steady state level of this mRNA increases as the cultures begin to form multilayered regions. High levels of the mRNA persist after the cells are trypsin-dissociated. Culture medium conditioned by nodular SMC also induces an increase of CLI mRNA. Analysis of RNA extracted from porcine tissues show the highest levels of CLI mRNA in brain and liver; lower levels are detected in other tissues, including the aorta. Possible functions for the CLI are discussed.

Regulation of collagenase and collagenase mRNA production in early- and late-passage human diploid fibroblasts.

The levels of collagenase and collagenase mRNA produced by early-passage (less than 40% of lifespan completed) and late-passage (greater than 80% of lifespan completed) cultures of human fibroblasts were analyzed. The constitutive levels of collagenase and collagenase mRNA produced by the late-passage cultures were 10-30 x greater than the levels observed in similarly treated early-passage cultures. Immunofluorescence analysis established that the percentage of collagenase-positive cells was also greater (77% vs. 4%) in the late-passage cultures. To determine whether the difference in collagenase production resulted from cell-derived regulatory factors, collagenase production was examined in cultures plated onto substrates coated with fibroblast extracellular matrix (ECM). Collagenase and collagenase mRNA production was enhanced in both types of cultures, although amounts produced by ECM-induced early-passage cultures was significantly less than that produced by similarly treated late-passage cultures. Collagen-coated substrates also induced collagenase synthesis.

Collagenase production by early and late passage cultures of human fibroblasts.

Comparison of the proteins secreted by early and late passage cell cultures of human fibroblasts revealed a high level of immunoreactive collagenase (Mr = 55,000 Da and 58,000 Da) in the late passage cell culture conditioned medium. Both molecular weight species reacted with a monoclonal anticollagenase antibody and were apparently glycosylation varaents of the same protein. The question of whether the apparent age-dependent differences in collagenase synthesis reflected changes in protein synthesis or secretion was addressed by assaying immunoreactive collagenase and collagenase mRNA. Immunofluorescence microscopy of cellular collagenase revealed that the percentage of collagenase positive cells ranged from 1 to 6% (early passage) to 35 to 46% (late passage) indicating that the late passage cells had higher basal levels of collagenase synthesis. Later passage cultures also secreted higher levels of immunoprecipitable collagenase into the culture medium and Northern analysis established that the basal level of collagenase mRNA was also 10 times greater in late passage cells. High basal levels of collagenase were also observed in fibroblasts cultured from an in vivo aged donor and from donors with Werner's syndrome. Collagenase production was induced in both early and late passage cell cultures by exposure to fibroblast extracellular matrix, fibroblast conditioned media, polypeptide growth factors, or phorbol esters. The induced levels were always greater in the late passage cell cultures than in the early passage cell cultures.