Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.     

Substratum influence on collagen and fibronectin biosynthesis by arterial smooth muscle cells in vitro

Collagen, fibronectin, and nonfibrous protein biosynthesis were examined in cultures of rabbit arterial smooth muscle cells grown on tissue culture plastic precoated either with rabbit plasma fibronectin or bovine serum albumin. Cells seeded into fibronectin-coated wells appeared to reach confluence more quickly than counterparts grown on albumin-coated surfaces. Measurement 3H-thymidine incorporation into DNA by these cultures suggested that this was probably a consequence of more rapid and efficient cell attachment rather than an increased rate of proliferation of smooth muscle cells grown on fibronectin. In preconfluent cultures, the rates of collagen and fibronectin biosynthesis were reduced to 34 and 57%, respectively, on a per-cell basis in cultures grown on fibronectin-coated surfaces compared with cells grown on albumin-coated plasticware. In preconfluent cultures grown on fibronectin-coated surfaces, a greater percentage of the total fibronectin synthesized was incorporated into the cell layer. The distribution of newly synthesized collagen between culture medium and cell layer, however, was not affected by alteration of substratum composition. There was no difference in the rate of synthesis of noncollagen proteins between the two groups of preconfluent cells. In postconfluent cultures the rates of collagen and fibronectin biosynthesis were equivalent in both albumin- and fibronectin-treated cultureware. In preconfluent cultures, analyses of procollagens showed that the overall amounts of both types I and III procollagens were reduced in fibronectin-treated wells, indicating the reduction in collagen synthesis to be general and not type-specific. Although type V procollagen biosynthesis was not detected in either preconfluent group, it was found in postconfluent cultures. The reduction of fibronectin synthesis in cells grown in fibronectin-coated wells was significant as early as 4 hours after plating. Together, these findings suggest that cultured arterial smooth muscle cells are capable of deriving information from their substratum and regulating the biosynthetic rates of extracellular matrix components in response to the immediate needs of the cell.     

Synthesis of types I and III procollagen and collagen by monkey aortic smooth muscle cells in vitro.

Analysis of pepsin-resistant proteins produced in culture by monkey aortic smooth muscle cells (SMC) indicates the synthesis of types I and III collagen. As determined by carboxymethylcellulose chromatography and disc gel electrophoresis, SMC cultures synthesize more type III collagen than monkey skin fibroblast cultures; aortic adventitial cell cultures (a mixture of SMC and fibroblasts) synthesize an intermediate amount of type III collagen. Both types I and III procollagens can also be isolated from the culture medium of SMC and skin fibroblasts. The procollagens were separated by diethylaminoethylcellulose (DEAE-cellulose) chromatography in identified by electrophoresis and after cleavage with pepsin and cyanogen bromide. Quantitation of the procollagen by DEAE-cellulose chromatography suggests that 68% of the SMC procollagens and less than 10% of the skin fibroblast procollagens are type III. On the other hand, estimation of the proportions of collagen types secreted by cells, employing pepsin digestion of cell culture medium at 15 degrees C, leads to an underestimation of the amount of type III collagen relative to type I. SMC and fibroblasts may differ in their ability to convert type I procollagen to collagen ad indicated by the observation that skin fibroblast culture medium contains both pN and pC collagen intermediates after 24 h, while cultures of SMC essentially lack the pC collagen intermediates.     

Procollagen mRNA metabolism during the fibroblast cell cycle and its synthesis in transformed cells.

Procollagen mRNA was isolated from mouse embryos and used for the synthesis of a highly labelled cDNA probe complementary to collagen mRNA. This probe was used for the investigation of procollagen mRNA metabolism during the cell cycle of 3T6 mouse embryo fibroblasts in culture. Titration hybridization experiments revealed that procollagen mRNA was present throughout the cell cycle following stumulation of confluent monolayers. Procollagen mRNA levels of sparse cultures appeared similar to those of unstimulated monolayers. The fluctuating levels of collagen synthesis during the cell cycle can be ascribed to changes in the amount of collagen mRNA present. In mouse sarcoma virus transformed 3T3 cells only 20--30% of the amount of procollagen mRNA in 3T3 cells is present indicating that the decline in collagen synthesis is due to mRNA availability.     

Differential effect of platelet-derived growth factor- versus serum-induced growth on smooth muscle alpha-actin and nonmuscle beta-actin mRNA expression in cultured rat aortic smooth muscle cells

Previous studies have demonstrated that rat aortic smooth muscle cells (SMC) show marked changes in smooth muscle (SM) alpha-actin content and fractional synthesis as a function of cell density and growth (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352; Blank, R., Thompson, M. M., and Owens, G. K. (1988) J. Cell Biol. 107, 299-306). Results of this study show that, although there is a 6-fold increase in SM alpha-actin content in postconfluent density arrested cultures as compared to proliferating subconfluent cultures, SM alpha-actin mRNA levels are not different between these cells. This suggests that the SM alpha-actin gene is constitutively active under both of these conditions and that accumulation of SM alpha-actin in postconfluent cells is due to translational and/or post-translational controls. The relationship between growth and cytodifferentiation was further explored by examining the effects of platelet-derived growth factor (PDGF)- or serum-induced growth on actin expression in postconfluent, quiescent cultures maintained in a defined serum-free media. Although both factors have been shown to stimulate proliferation and decrease fractional SM alpha-actin synthesis (Blank et al., 1988), their effects on actin mRNA levels were quite different. PDGF was found to induce a dramatic drop in SM alpha-actin steady state mRNA level but had no effect on nonmuscle beta-actin mRNA level. In contrast, serum stimulation was shown to increase nonmuscle beta-actin mRNA level, whereas SM alpha-actin mRNA level remained constant. Taken together these results indicate that PDGF is a specific and potent repressor of SM alpha-actin expression in vascular SMC and implicate a possible developmental role for PDGF in control of SMC differentiation. In addition, the observation that the level of SM alpha-actin mRNA is unaltered in serum-stimulated cells indicates that an absolute decrease in SM alpha-actin mRNA is not obligatory for cell cycle entrance.     

Influence of cell density on collagen biosynthesis in fibroblast cultures.

Characteristic features of collagen metabolism in human skin fibroblasts were studied in relation to cell density. Measuring peptide-bound hydroxyproline we found that collagen synthesis per cell decreased when cultures approached confluency. On the other hand, the relative rate of collagen synthesis (collagen/total protein) was higher in quiescent than in proliferating cultures. With increasing cell density the proportion of type III collagen in comparison with type I was found to be slightly increased. In addition, in low-density cultures [alpha I(I)]3 collagen trimers were produced in considerable amounts, whereas they were no longer detected in cultures with a high cell density. Although hydroxylation of proline residues was normal in all cell stages, conversion of procollagen into collagen was found to depend strongly on the density at which the cells were investigated. Almost no cleavage of procollagen peptides was observed in rapidly growing cells, whereas highly confluent cell cultures converted most of the newly synthesized procollagen molecules.     

Collagen synthesis by monkey arterial smooth muscle cells during proliferation and quiescence in culture

Monkey arterial smooth muscle cells (SMC) which are stimulated to proliferate in the presence of 5% monkey blood serum (MBS) and which remain quiescent in 5% monkey platelet-poor plasma serum (MPPPS) were examined for their ability to synthesize collagen in each of these conditions in culture. Collagen synthesis was measured by determining amounts of newly formed labeled hydroxyproline, following labelling in the presence of [³H]proline and ascorbic acid. Ascorbate requirements of SMC were examined to assure maximal hydroxylation. SMC synthesize the same amount of collagen/cell in 5% whole blood serum (MBS) during the early phase of rapid proliferation as during slow growth in later phases in culture. SMC grown in the presence of serum-lacking platelet factors synthesize 60–90% less collagen and 60–90% less non-collagen protein (per cell or per mg protein) than cells grown in MBS. Non-collagen protein synthesis was measured as incorporation of both [³H]proline and of [³H]leucine, determined as trichloroacetic acid (TCA)-precipitable material. Previous studies indicate that a factor derived from platelets is the principal mitogen present in whole blood serum for diploid cells such as SMC and fibroblasts in culture. Similarly derived factors are potent stimulators of both collagen and non-collagen protein synthesis by SMC. SMC, quiescent in medium lacking platelet derived material (MPPPS), is being used to investigate factors important in SMC proliferation since this is a significant event in atherogenesis in vivo. An increased deposition of collagen also occurs during atherogenesis. Consequently it will be useful to employ similar cultures of quiescent SMC to examine agents which affect production of this connective tissue matrix protein.     

Palmitoyl ascorbate: selective augmentation of procollagen mRNA expression compared with L-ascorbate in human intestinal smooth muscle cells.

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of L-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 microM. By contrast, L-ascorbate was required at 4-5 times the concentration for the same response. However, at 20 microM, both palmitoyl and L-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas L-ascorbate had no effect. Palmitoyl ascorbate and L-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or L-ascorbate on the activity of a procollagen alpha2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than L-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro.     

Regulation of alpha-smooth muscle actin and other polypeptides in proliferating and density-arrested vascular smooth muscle cells

We have examined alpha-smooth muscle actin (alpha-SM actin) protein and mRNA levels in proliferating and density-arrested rabbit vascular smooth muscle cells (SMC) and also studied overall polypeptide synthesis in these cells by two-dimensional (2-D) gel electrophoresis. Of the approximately 1,000 cellular polypeptides resolved by 2-D gel analysis, we consistently detected increased expression of 12 polypeptides in growth-arrested SMC. These polypeptides, with apparent molecular weights of 24,000 to 55,000 exhibited relative increases of between fourfold to greater than tenfold. Three of these polypeptides were expressed at undetectable levels in proliferating SMC. We also detected 12 secreted polypeptides that were expressed at higher levels in growth-arrested SMC. More changes were associated with the secreted polypeptides, since they represented approximately 4% of the total resolved secreted polypeptides, while only 1% of the cellular polypeptides were increased in high-density growth-arrested cells. Under these conditions we observed no change in relative alpha-SM actin protein content as determined by 2-D gel analysis and Western blots. This was corroborated by high levels of alpha-SM actin mRNA levels in both proliferating and high-density growth-arrested SMC. These results indicate rabbit vascular SMC maintain a high level of expression of a smooth muscle differentiation marker (alpha-SM actin) in a proliferation- and density-independent manner. We also examined polypeptide synthesis in SMC isolated by enzymatic digestion of the aorta vs. cells isolated by the explant method. We found that although overall protein patterns were remarkably similar, several differences were observed. These differences were not due to increased contamination by fibroblasts, since both enzymatically- and explant-derived SMC contained high levels of alpha-SM actin as determined by immunofluorescence and by Northern analysis.     

Enhanced cell accumulation and collagen processing by keratocytes cultured under agarose and in media containing IGF-I,TGF-β or PDGF

We previously showed an agarose overlay on keratocytes cultured in media containing pharmacological levels of insulin enhanced collagen processing and collagen fibril formation. In this study, we compared collagen processing by keratocytes cultured in media containing physiological levels of IGF-I, TGF-β, FGF-2, and PDGF in standard and in agarose overlay cultures. Pepsin digestion/SDS PAGE was used to determine the levels of procollagen secreted into the media and the collagen content of the ECM associated with the cell layer. Distribution of collagen type I and fibronectin in the ECM of the agarose cultures was determined by immunoflorescence. Collagen fibril and keratocyte morphology was evaluated by electron microscopy. The agarose overlay significantly enhanced the cell number in the IGF-I, TGF-β and PDGF treated cultures by 2–3 fold. The overlay also significantly enhanced the processing of procollagen to collagen fibrils from 29% in standard cultures to 63–68% in agarose cultures for the IGF-I and PDGF cultures, and from 66% in standard culture to 85% in agarose culture for the TGF-β cultures. Cell accumulation and collagen processing was not enhanced by agarose overlay of the FGF-2 treated cultures. Collagen type I and fibronectin were more uniformly distributed and the collagen fibrils smaller in the ECM of the TGF-β treated cultures. Keratocytes in the FGF-2 treated cultures were in close cell contact with few collagen fibrils while IGF-I, TGF-β, and PDGF cultures had an extensive ECM with abundant collagen fibrils. The results of this study indicate that the agarose overlay enhances collagen fibril assembly and cell accumulation by keratocytes when both collagen synthesis and cell proliferation are stimulated.     

Expression of smooth muscle-specific alpha-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation

The relationship between growth and cytodifferentiation was studied in cultured rat aortic smooth muscle cells (SMCs) using expression of the smooth muscle (SM)-specific isoactins (Vanderkerckhove, J., and K. Weber, 1979, Differentiation, 14:123-133) as a marker for differentiation in these cells. Isoactin expression was evaluated by: (a) measurements of fractional isoactin content and synthesis ([35S]methionine incorporation) by densitometric evaluation of two-dimensional isoelectric focusing sodium dodecyl sulfate gels, and (b) immunocytological examination using SM-specific isoactin antibodies. Results showed the following: (a) Loss of alpha-SM isoactin was not a prerequisite for initiation of cellular proliferation in primary cultures of rat aortic SMCs. (b) alpha-SM isoactin synthesis and content were low in subconfluent log phase growth cells but increased nearly threefold in density-arrested postconfluent cells. Conversely, beta-nonmuscle actin synthesis and content were higher in rapidly dividing subconfluent cultures than in quiescent postconfluent cultures. These changes were observed in primary and subpassaged cultures. (c) alpha-SM actin synthesis was increased by growth arrest of sparse cultures in serum-free medium (SFM; Libby, P., and K. V. O'Brien, 1983, J. Cell. Physiol., 115:217-223) but reached levels equivalent to density-arrested cells only after extended periods in SFM (i.e., greater than 5 d). (d) SFM did not further augment alpha-SM actin synthesis in postconfluent SMC cultures. (e) Serum stimulation of cells that had been growth-arrested in SFM resulted in a dramatic decrease in alpha-SM actin synthesis that preceded the onset of cellular proliferation. These findings demonstrate that cultured vascular SMCs undergo differential expression of isoactins in relation to their growth state and indicate that growth arrest promotes cytodifferentiation in these cells.     

Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells

Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.     

Mechanical Load Enhances Procollagen Processing in Dermal Fibroblasts by Regulating Levels of Procollagen C-Proteinase

Mechanical forces are emerging as key regulators of cell function. We hypothesize that mechanical load may influence dermal fibroblast activity. We assessed the direct effects of mechanical load on human dermal fibroblast procollagen synthesis and processing in vitro. Cells were loaded in a biaxial loading system (Flexercell 3000). Hydroxyproline levels were measured in the medium and cell layer as an estimate of procollagen synthesis and processing to insoluble collagen. Mechanical load (in the presence of serum or TGF-beta) enhanced procollagen synthesis by 45 +/- 3% (P < 0.001), and 38 +/- 4% (P < 0.001), respectively, over unloaded growth factor controls after 48 h. Insoluble collagen deposition was enhanced in the same cultures by 115 +/- 8% (P < 0.01) and 72% +/- 9% (P < 0.01), respectively. This effect was inhibited using l-arginine suggesting that procollagen C-proteinase, the enzyme which directly cleaves the C-terminal propeptide of procollagen to form insoluble collagen, is required for the fiber formation observed. Procollagen mRNA levels in loaded samples increased by more than two-fold in both serum and TGF-beta-treated cultures at 48 h. Procollagen C-proteinase mRNA levels were also enhanced by a similar magnitude, although the increase was observed at 24 h. Procollagen C-proteinase protein levels were also increased at this time. Protein and mRNA levels of the procollagen C-proteinase enhancer protein, which binds the C-terminal propeptide of procollagen to enhance the rate of peptide cleavage, were unaffected by mechanical load. This study demonstrates that mechanical load promotes procollagen synthesis in dermal fibroblasts by enhancing gene expression and posttranslational processing of procollagen.     

Altered glycosaminoglycan production in cultured osteogenesis-imperfecta skin fibroblasts.

Collagen and glycosaminoglycan syntheses were studied in skin fibroblasts cultured from patients with osteogenesis imperfecta (OI) and from age-matched controls. Collagen synthesis (measured as protein-bound [3H]hydroxyproline) was decreased in all four OI cell lines studied in the present experiments, comprising 16-24% of total protein synthesis (40% in normal cells). Hyaluronic acid production in OI skin fibroblasts per cell was higher than in age-matched controls, but the production of sulphated glycosaminoglycans was at the normal level. Thus the ratio of the hyaluronic acid and sulphated-glycosaminoglycan radioactivities was markedly higher in OI cultures than in control cultures, especially at the exponential phase of growth where the synthesis of hyaluronic acid was highest. Hyaluronic acid in OI had a normal molecular weight when determined by gel filtration on Sepharose 2B. The removal of high-molecular-weight hyaluronic acid from the medium by hyaluronidase had no effect on the rate of collagen secretion in OI cell line 1 (A.T.C.C. 1262), in which the rate of collagen secretion was lowest.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

Evidence for pretranslational regulation of collagen synthesis by procollagen propeptides

We present, here, evidence for a pretranslational role of procollagen propeptides in the regulation of collagen synthesis. Amino- and carboxyl-terminal type I procollagen propeptides were isolated and purified from chick calvaria and tendon cultures. Human lung fibroblasts (IMR-90) were incubated in medium containing varying concentrations of propeptides. Amino-propeptides at 10 nM caused an 80% decrease in collagen synthesis compared to control. Higher concentrations of amino-propeptides did not decrease collagen synthesis further and no significant effect on non-collagen synthesis was found throughout the entire concentration range. Carboxyl-propeptides also inhibited collagen synthesis. At 10 nM, collagen synthesis was decreased by 30% and a concentration of 40 nM caused an 80% reduction. However, at the latter concentration non-collagen synthesis was also affected, decreasing by 20% relative to control. To assess possible pretranslational effects of propeptides, IMR-90 fibroblasts were treated with varying concentrations of each propeptide and levels of type I procollagen mRNA was determined by dot hybridization with a 32P-alpha 2(I) cDNA probe. Both propeptides caused significant concentration-dependent decreases in procollagen type I mRNA levels. At 10 nM, the amino-propeptide resulted in a 55% decrease in collagen mRNA levels while at 40 nM these levels decreased by 72% compared to control. Carboxyl-propeptides were also inhibitory, decreasing mRNA levels by 33% at 10 nM and 73% at 40 nM. Messenger RNA levels of a representative noncollagenous protein, beta-actin, were unaffected by either propeptide throughout the concentration range.     

1,25-Dihydroxyvitamin D3 interaction with dexamethasone and retinoic acid: effects on procollagen messenger ribonucleic acid levels in rat osteoblast-like cells

We previously have reported that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], dexamethasone, and retinoic acid inhibit collagen synthesis in rat osteoblast-like cell primary cultures. We also have found that dexamethasone increases 1,25-(OH)2D3 receptor levels in these cells. Furthermore, this increase in 1,25-(OH)2D3 receptor level is paralleled by an enhanced inhibition of collagen synthesis when dexamethasone and 1,25-(OH)2D3 are used in combination. In contrast, retinoic acid at high doses decreases 1,25-(OH)2D3 receptor level in rat osteoblast-like cells and attenuates 1,25-(OH)2D3 inhibition of collagen synthesis. In the present study, we have used a [32P]cDNA probe for rat pro alpha 1 (I) to determine if these osteotropic agents act by modulating steady state procollagen mRNA levels. Hybridization with a [32P]cDNA probe for human actin was used as a control. We find that the steady state levels of procollagen mRNA are decreased in all cases, while there are negligible changes in actin mRNA levels. Dexamethasone, at the low dose of 13 nM, acts synergistically with 1,25-(OH)2D3 in decreasing procollagen mRNA levels. The effects of retinoic acid and 1,25-(OH)2D3 are additive at low doses (13 and 130 nM); however, at a high dose of retinoic acid (1.3 microM), combined treatment with 1,25-(OH)2D3 does not reduce procollagen mRNA levels beyond the decrease due to retinoic acid alone. The reduction in procollagen mRNA level after each of these treatments falls in the same range as inhibition of collagen synthesis measured at the protein level. These data suggest that the synthesis of collagen under these treatments is controlled primarily through modulation of steady state procollagen mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of type I collagen genes in cultured scleroderma fibroblasts

Fibroblasts cultured from affected skin areas of five patients with cutaneous scleroderma were found to produce increased amounts of collagen when compared with nonaffected control cells. Total RNA was isolated from the cultures and analyzed for its level of pro alpha 1 (I)collagen mRNA by hybridization of RNA blots with a cloned cDNA probe. The levels of pro alpha 1 (I)collagen mRNAs relative to total RNA were two- to sixfold higher in the samples from affected cells, accounting for the increased synthesis of type I collagen. Cytoplasmic dot hybridizations were performed to measure the cellular content of pro alpha 1 (I)collagen mRNA: up to ninefold increases in the level of this mRNA per cell were found. Upon subculturing, scleroderma fibroblasts were found to reduce gradually the increased synthesis of collagen to the level of nonaffected controls by the tenth passage. The levels of type I collagen mRNAs were also reduced, but more slowly. The results suggest that in scleroderma fibroblasts the genes for type I collagen are activated at procollagen mRNA level or that they are more stable and that the activating factors are lost during prolonged cell culture because cells from affected areas lose their activated state.