Post-transcriptional regulation of the pro alpha 1(I) collagen gene in pro alpha 1(I)-deficient, chemically transformed Syrian hamster embryo fibroblasts.

4-Nitroquinoline-1-oxide-transformed Syrian hamster embryo fibroblasts (NQT-SHE) synthesize the pro alpha 2 chain but not the pro alpha 1 subunit of type I procollagen, and they contain little pro alpha 1(I)mRNA. This study shows that there was no accumulation of pro alpha 1(I) poly(A)+ mRNA in NQT-SHE fibroblasts. BHK cells, a normal established line of hamster fibroblasts that synthesized collagen at approximately the same rate as NQT-SHE fibroblasts, nevertheless produced both subunits of type I collagen and contained pro alpha 1(I)mRNA. Run-off transcription assays with isolated nuclei showed that both the pro alpha 1(I) and pro alpha 2(I) genes were transcribed at about the same rate in NQT-SHE cells as well as in the normal BHK cells. These results suggest that a post-transcriptional defect, probably resulting from transformation, prevents the accumulation of pro alpha 1(I)mRNA in NQT-SHE cells.

Scorbutic and fasted guinea pig sera contain an insulin-like growth factor I-reversible inhibitor of proteoglycan and collagen synthesis in chick embryo chondrocytes and adult human skin fibroblasts.

Chick embryo chondrocytes cultured in sera from scorbutic and fasted guinea pigs exhibited decreases in collagen and proteoglycan production to about 30-50% of control values (I. Oyamada et al., 1988, Biochem. Biophys. Res. Commun. 152, 1490-1496). Here we show by pulse-chase labeling experiments that in the chondrocyte system, as in the cartilage of scorbutic and fasted guinea pigs, decreased incorporation of precursor into collagen was due to decreased synthesis rather than to increased degradation. There was a concomitant decrease in type II procollagen mRNA to about 32% of the control level. As in scorbutic cartilage, proteoglycan synthesis by chondrocytes in scorbutic serum was blocked at the stage of glycosaminoglycan chain initiation. Scorbutic and fasted guinea pig sera also caused a 50-60% decrease in the rates of collagen and proteoglycan synthesis in adult human skin fibroblasts, which synthesize mainly type I collagen. Decreased matrix synthesis in both cell types resulted from the presence of an inhibitor in scorbutic and fasted sera. Elevated cortisol levels in these sera were not responsible for inhibition, as determined by the addition of dexamethasone to chondrocytes cultured in normal serum. Insulin-like growth factor I (IGF-I, 300-350 ng/ml) reversed the inhibition of extracellular matrix synthesis by scorbutic and fasted guinea pig sera in both cell types and prevented the decrease in type II procollagen mRNA in chondrocytes. Therefore, in addition to its established role in proteoglycan metabolism, IGF-I also regulates the synthesis of several collagen types. An increase in the circulating inhibitor of IGF-I action thus could lead to the negative regulation of collagen and cartilage proteoglycan synthesis that occurs in ascorbate-deficient and fasted guinea pigs.

Effect of synthetic human parathyroid hormone on the levels of alkaline phosphatase activity and formation of alkaline phosphatase-rich matrix vesicles by primary cultures of chicken epiphyseal growth plate chondrocytes.

The effect of synthetic human parathyroid hormone (hPTH) on the formation of matrix vesicles (MV), and on the rate of cell division, production of cellular alkaline phosphatase (AP) and protein by primary cultures of chicken epiphyseal growth plate hypertrophic chondrocytes was investigated. Addition to serum-containing or serum-free media of physiological levels of hPTH, in a range from 0.1 to 10 nM, caused a progressive decrease in the formation of AP-rich MV. However, studies on incorporation of [3H]choline into MV indicate that MV formation per se was not significantly decreased. hPTH was found to markedly decrease the expression of cellular AP, accompanied by an increase in cell division [( 3H]thymidine incorporation) and protein synthesis. Since these effects of hPTH were augmented by 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, and mimicked by the cAMP analogue N6,O2'-dibutyryl-adenosine 3',5'-cyclic-monophosphate (DBcAMP), the findings clearly indicate that hPTH was acting through the classic cAMP-mediated mechanism. Inasmuch as elevation of AP in growth plate chondrocytes coincides with MV formation, maturation and hypertrophy of the cells, and induction of mineralization, the stimulation of cell division and suppression of cellular AP indicates that hPTH would cause the cells to revert to a less differentiated state. Thus, elevation in PTH, which results from lowered circulating levels of Ca2+, should inhibit mineral deposition in the growth plate. This may be a physiological protective mechanism to prevent a further drain on serum Ca2+.

Effect of trifluoperazine and other drugs on matrix vesicle formation by chicken growth plate chondrocytes in primary cell culture.

Growth plate chondrocytes in primary culture release alkaline phosphatase (AP)-rich matrix vesicles (MV) into the culture medium. While these are thought to derive from the plasma membrane by a membrane fusion-dependent process, the mechanism is not fully understood. Recently, a cytosolic protein, synexin, has been shown to promote membrane fusion in a number of systems, and thus may be involved in MV formation. Since the action of synexin is selectively inhibited by trifluoperazine (TFP) and other phenothiazines, we examined the effects of these drugs, and imipramine, on cellular AP production and formation of AP-containing MV by cultured chondrocytes. In addition, we studied the effect on cell division, protein biosynthesis, and incorporation of palmitate into cellular and MV lipids. All of the drugs reduced cellular AP in a concentration-dependent manner; however, at the higher levels, chlorpromazine (CPZ) and TFP caused a transient sharp rise in MV AP activity. At IC50 levels, the drugs were more inhibitory to cellular AP than to MV AP, appearing to enhance significantly the transfer of available cellular AP into MV. In contrast, the drugs stimulated incorporation of [3H]palmitate into cellular lipids, but either had no effect on, or actually inhibited, incorporation of the fatty acid into MV. At these levels, the drugs had little effect on cell division and protein biosynthesis. The inhibitory effect of IC50 levels of CPZ on palmitate incorporation into MV appears to have resulted from impairment of vesicle formation per se, since at these levels the drug stimulated incorporation of the fatty acid into the cells. The transient stimulatory effect of higher levels of CPZ on MV AP levels, and the enhanced transfer of AP into MV by the drugs generally, may result from the effect of the drugs on membrane structure. Since TFP was not inhibitory to MV formation, it is doubtful that synexin was directly involved.

Effect of amino acid levels on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture.

The effect of varying the amino acid concentrations of the culture medium on matrix vesicle formation was studied in primary cultures of chicken epiphyseal growth plate chondrocytes grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS). Decreasing the levels of free amino acids in the culture medium to levels of one-half, one quarter, and one eighth of the values normally present in DME caused a progressive decline in matrix vesicle (MV) formation. Increasing the level in the culture medium of those amino acids that are enriched in extracellular fluid (ECF) of growth plate cartilage significantly increased formation of matrix vesicles (MV), as assayed by the alkaline phosphatase (AP) activities present in high-speed sediments from spent culture media. However, adjusting the levels of all amino acids to match those of the ECF produced the greatest stimulation of MV formation. Of the amino acids that are notably enriched in ECF, glutamate (GLU), alanine (ALA), serine (SER), asparagine (ASN), and taurine (TAU) individually enhanced MV production, whereas proline (PRO), glycine (GLY), and aspartate (ASP) had essentially no effect. The simple combination of ECF levels of ALA and GLU resulted in a stimulation of MV formation equal to that observed when the eight aforementioned amino acids were elevated to ECF levels. Other combinations of ASP and GLY, or of TAU, SER, and ASN showed some stimulation, but at a lower level. Increasing the amino acid concentrations, alone or in combination, also increased the levels of cellular AP, and to a lesser extent cellular protein. While increases in cellular AP were generally correlated with increased formation of AP-rich MV, this was not uniformly true. These results indicate that in addition to hormones and growth factors, nutritional factors such as the levels of amino acids are also critical for normal phenotypic expression, growth, and matrix formation by epiphyseal chondrocytes.