Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells

The role of ascorbate in the production and secretion of procollagen by human intestinal smooth muscle cells and the conditions in culture for optimal ascorbate bioefficacy were studied. Procollagen synthesis and secretion were determined by the incubation of cells with L-[5-³H]proline, and the quantitation of radiolabelled procollagen bands in the cell layer and the culture medium by polycrylamide slab gel electrophoresis and densitometry. When cells were cultured without ascorbate in the culture medium, procollagen secretion into the medium was 75% less than in cells receiving fresh ascorbate daily. In the cell layer, in contrast, procollagen accumulation was fourfold greater in the scorbutic cells than in the ascorbate-replete cells. These findings contrasted with those in a control line of scorbutic human dermal fibroblasts in which a 95% decrease in procollagen secretion was not associated with any procollagen accumulation in the cells. In the intestinal smooth muscle cells, the absence of ascorbate resulted in a 25 and 50% decrease in steady-state levels of procollagen I and III mRNA, respectively, compared to a 40 and 75% decrease in fibroblasts. Heat inactivation of the serum in the culture medium augmented the promotion of procollagen secretion by ascorbate two- to fourfold. L-ascorbate phosphate did not increase the activity of L-ascorbate when replaced in medium either daily or every 4 days, and its efficacy was not augmented by serum heat inactivation. The changing of culture medium induced collagen secretion in the absence of ascorbate, but this process was markedly enhanced by ascorbate and induced a transient decrease in the steady-state levels of both procollagen and nonprocollagen mRNAs. The predominant action of L-ascorbate on HISM cells in vitro is to promote procollagen secretion and not procollagen synthesis. L-ascorbate-phosphate is not an adequate substitute for L-ascorbate in this cell line. © 1995 Wiley-Liss, Inc.     

Role of procollagen mRNA levels in controlling the rate of procollagen synthesis.

Two factors must be present for primary avian tendon cells to commit 50% of their total protein production to procollagen: ascorbate and high cell density. Scorbutic primary avian tendon cells at high cell density (greater than 4 X 10(4) cells per cm2) responded to the addition of ascorbate by a sixfold increase in the rate of procollagen synthesis. The kinetics were biphasic, showing a slow increase during the first 12 h followed by a more rapid rise to a maximum after 36 to 48 h. In contrast, after ascorbate addition, the level of accumulated cytoplasmic procollagen mRNA (alpha 2) showed a 12-h lag followed by a slow linear increase requiring 60 to 72 h to reach full induction. At all stages of the induction process, the relative increase in the rate of procollagen synthesis over the uninduced state exceeded the relative increase in the accumulation of procollagen mRNA. A similar delay in mRNA induction was observed when the cells were grown in an ascorbate-containing medium but the cell density was allowed to increase. In all cases, the rate of procollagen synthesis peaked approximately 24 h before the maximum accumulation of procollagen mRNA. The kinetics for the increase in procollagen synthesis are not, therefore, in agreement with the simple model that mRNA levels are the rate-limiting factor in the collagen pathway. We propose that the primary control point is at a later step. Further support for this idea comes from inhibitor studies, using alpha, alpha'-dipyridyl to block ascorbate action. In the presence of 0.3 mM alpha, alpha'-dipyridyl there was a specific two- to threefold decrease in procollagen production after 4 h, but this was unaccompanied by a drop in procollagen mRNA levels. Therefore, inhibitor studies give further support to the idea that primary action of ascorbate is to release a post-translational block.     

Procollagen secretion meets the minimum requirements for the rate-controlling step in the ascorbate induction of procollagen synthesis

Ascorbate addition to primary avian tendon cells has been shown previously to cause a approximately 6-fold increase in procollagen translation that is first observable after 4 h and reaches a maximum level after 48 h. Similarly, procollagen mRNA has been shown to increase after ascorbate addition by approximately 6-fold starting at 12 h and reaching a maximum level by 72 h. The rate constant for procollagen secretion is now shown to also react to ascorbate by a 6-fold change. This results in a drop in the half-life of procollagen within the cell from 120 to 20 min. In sharp contrast to the other steps in the procollagen pathway, the change in the secretion rate constant is extremely fast occurring in less than 30 min. Moreover, after ascorbate addition, greater than 80% of the internal procollagen can be secreted at the fast rate. Since this change results from an increase in hydroxylation of proline residues and since the hydroxylation reaction has been localized to the endoplasmic reticulum, this evidence strongly supports the model that the slow step in the secretion pathway is transport out of the endoplasmic reticulum. Further support for this comes from electron microscope autoradiography of [3H]proline-labeled cells where the labeled procollagen pool within the cells was highly localized to the endoplasmic reticulum.     

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.     

Ascorbate induction of collagen synthesis as a means for elucidating a mechanism of quantitative control of tissue-specific function.

Ascorbic acid displays the characteristics of an ideal inducer of tissue-specific function in primary avian tendon cells in culture. It is a highly specific, potent stimulator of collagen synthesis, it demonstrates slow reversible kinetics, and it has no effect on growth rate of the cultured cells. Kinetic analysis of ascorbate induction of collagen synthesis was used to determine the critical steps in this complex biosynthetic pathway. Full hydroxylation of the proline residues in collagen, although probably a necessary step for collagen induction, was in itself not sufficient for achieving either increased secretion or increased synthesis. On the other hand, an increase in secretion rate, which required both the presence of ascorbate and a high cell density, did correlate with the later stimulation in procollagen production. The process of procollagen secretion, therefore, meets the minimal requirements for the rate-limiting step. The fact that the cells maintained a large pool of intracellular procollagen despite changes in the rates of translation or secretion led us to postulate a possible feedback between the level of the internal procollagen pool and the rate of procollagen synthesis.     

Decreased extracellular matrix production in scurvy involves a humoral factor other than ascorbate

Our recent studies suggested that decreased collagen synthesis in bone and cartilage of scorbutic guinea pigs was not related to ascorbate-dependent proline hydroxylation. The decrease paralleled scurvy-induced weight loss and reduced proteoglycan synthesis. Those results led us to propose that the effects of ascorbate deficiency on extracellular matrix synthesis were caused by changes in humoral factors similar to those that occur in fasting. Here we present evidence for this proposal. Exposure of chick embryo chondrocytes to scorbutic guinea pig serum, in the presence of ascorbate, led to effects on extracellular matrix synthesis similar to those seen in scorbutic animals. The rates of collagen and proteoglycan synthesis were reduced to approximately 30-50% of the levels in cells cultured in normal guinea pig serum plus ascorbate, but proline hydroxylation and procollagen secretion were unaffected. Similar results were obtained with serum from fasted guinea pigs supplemented in vivo with ascorbate. The growth rate of the chondrocytes was not significantly affected by scorbutic guinea pig serum.     

Mechanisms of stimulation of collagen synthesis during chick embryonic skin development.

To study how collagen synthesis is regulated in developing chick embryonic skin, hydroxyproline synthesis, incorporation of proline, and translational activity and content of collagen mRNA in 12-, 15-, and 18-day skins were determined and compared with each other. Hydroxyproline synthesis in the 18-day skins was markedly increased over that in the 12-day skins, whereas proline incorporation was moderately increased. The increase in collagen synthesis from day 15 to 18 was accompanied by increases in both the translational activity and the content of type I procollagen mRNA, with a selective increase in the lower-molecular-weight species of pro alpha 1 (I) collagen mRNA. In contrast, the stimulation of collagen synthesis from day 12 to day 15 did not parallel the levels of type I procollagen mRNA. These results suggest that the stimulation of collagen synthesis is regulated by collagen mRNA levels only in the later stage of development (from day 15 to day 18). Both the collagen synthesis and type I procollagen mRNA levels in the fibroblasts isolated on each corresponding day were constant. The difference in collagen synthesis under two different culture conditions suggests that cell-matrix interaction and/or some serum factors, including several growth factors, are essential for the marked stimulation of collagen synthesis observed in 12- to 18-day skin.     

Regulation of procollagen synthesis during the development of chick embryo calvaria. Correlation with procollagen mRNA content

During the embryonic development of chick calvaria (membranous cranial bones), the relative rate of procollagen synthesis increased from about 12% of total protein synthesis on Day 10 to about 65% on Day 17. This increase is due to a 1.7-fold increase in the absolute rate of procollagen synthesis and a 3-fold decrease in the synthesis of noncollagenous proteins. The increase in procollagen synthesis is directly proportional to an increase in procollagen mRNA content per cell as measured either by cell-free translation or by hybridization with complementary DNA. The results indicate that translational control of procollagen mRNA does not play a substantial role during calvaria development and that the specialization in the synthesis of this protein is largely due to the loss or inactivation of mRNAs for noncollagenous proteins.     

Heparin increases mRNA levels of thrombospondin but not fibronectin in human vascular smooth muscle cells

The effects of heparin (180 micrograms/ml) on steady state mRNA levels for fibronectin, thrombospondin, actin and collagen types I and III were investigated in human umbilical artery smooth muscle cells. Heparin caused a 120% increase in thrombospondin mRNA levels and a 60% and 180% increase in the mRNA levels of procollagen chains alpha 2(I) and alpha 1(III), respectively. No change in fibronectin or actin mRNA levels resulted from heparin treatment. We reported earlier (Biochem. Biophys. Res. Comm. 148:1264, 1987) that heparin increases smooth muscle cell synthesis of both fibronectin and thrombospondin. These data show that heparin coordinately regulates thrombospondin mRNA and protein levels. The heparin induced increase in fibronectin biosynthesis apparently reflects control at the translational or post-translational level.     

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.     

Prolyl hydroxylase production can be uncoupled from the regulation of procollagen synthesis

Primary avian tendon (PAT) cells increase the production of procollagen from 10-12% to 40-50% of total protein synthesis in response to the addition of ascorbate and an increasing cell density. We now show that prolyl hydroxylase (PH) also increases its activity by greater than five-fold in response to increasing cell density; but unlike procollagen production, this is independent of the presence of ascorbate. The increased activity is a result of greater enzyme production and not a shift in the ratio of inactive to active forms which remains constant at about 10% of the total enzyme proteins. We present the possibility that at low cell density the levels of PH activity could limit production of collagen.     

Glucose-induced translational control of proinsulin biosynthesis is proportional to preproinsulin mRNA levels in islet beta-cells but not regulated via a positive feedback of secreted insulin

Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (/=10-fold). Importantly, neither exogenously added nor secreted insulin were found to play any role in regulating insulin secretion, proinsulin translation, preproinsulin mRNA levels, or total protein synthesis. The results presented here indicate that long term nutritional state sets the preproinsulin mRNA level in the beta-cell at which translation control regulates short term changes in rates of proinsulin biosynthesis in response to glucose, but this is not mediated by any autocrine effect of insulin.     

Ethyl-3,4-dihydroxybenzoate inhibits myoblast differentiation: evidence for an essential role of collagen

To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with alpha-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pro alpha 1(I) and pro alpha 2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.     

Regulation of ribosomal protein mRNA content and translation in growth-stimulated mouse fibroblasts

When resting (G₀) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates of synthesis of rRNA and ribosomal proteins increase, resulting in an increase in ribosome content beginning about 6 h after stimulation. In this study, we monitored the content, metabolism, and translation of ribosomal protein mRNA (rp mRNA) in resting, exponentially growing, and serum-stimulated 3T6 cells. Cloned cDNAs for seven rp mRNAs were used in DNA-excess filter hybridization studies to assay rp mRNA. We found that about 85% of rp mRNA is polyadenylated under all growth conditions. The rate of labeling of rp mRNA relative to total polyadenylated mRNA changed very little after stimulation. The half-life of rp mRNA was about 11 h in resting cells and about 8 h in exponentially growing cells, values which are similar to the half-lives of total mRNA in resting and growing cells (about 9 h). The content of rp mRNA relative to total mRNA was about the same in resting and growing 3T6 cells. Furthermore, the total amount of rp mRNA did not begin to increase until about 6 h after stimulation. Since an increase in rp mRNA content did not appear to be responsible for the increase in ribosomal protein synthesis, we determined the efficiency of translation of rp mRNA under different conditions. We found that about 85% of pulse-labeled rp mRNA was associated with polysomes in exponentially growing cells. In resting cells, however, only about half was associated with polysomes, and about 30% was found in the monosomal fraction. The distribution shifted to that found in growing cells within 3 h after serum stimulation. Similar results were obtained when cells were labeled for 10.5 h. About 70% of total polyadenylated mRNA was in the polysome fraction in all growth states regardless of labeling time, indicating that the shift in mRNA distribution was species specific. These results indicate that the content and metabolism of rp mRNA do not change significantly after growth stimulation. The rate of ribosomal protein synthesis appears to be controlled during the resting-growing transition by an alteration of the efficiency of translation of rp mRNA, possibly at the level of protein synthesis initiation.