Regulation of collagen biosynthesis by ascorbic acid: a review.

L-ascorbic acid is an essential cofactor for lysyl hydroxylase and prolyl hydroxylase, enzymes essential for collagen biosynthesis. In addition, L-ascorbic acid preferentially stimulates collagen synthesis in a manner which appears unrelated to the effect of L-ascorbic acid on hydroxylation reactions. This reaction is stereospecific and unrelated to intracellular degradation of collagen. The effect apparently occurs at a transcriptional or translational level, since L-ascorbic acid preferentially stimulates collagen-specific mRNA. In addition, it stimulates lysyl hydroxylase activity but inhibits prolyl hydroxylase activity in human skin fibroblasts in culture.

Stimulation by glutamine of the formation of N6-hydroxylysine in a cell-free extract from Aerobacter aerogenes 62-1.

Glutamine may serve as an activator and/or regulator of the N6-hydroxylase (E.C. 1.14.99) of Aerobacter aerogenes 62-1. Activation and stabilization of N6-hydroxylase activity was observed both in vivo and in vitro. Growth in a glutamine-supplemented medium resulted in (1) maximum N6-hydroxylase activity at an earlier stage of growth and (2) higher N6-hydroxylase activity and continued aerobactin synthesis into stationary phase. Storage of P2 in the presence of L-glutamine (1 mM) significantly increased the lifetime of the labile N6-hydroxylase activity. Inclusion of L-glutamine in the incubation mixture typically resulted in a 2-3-fold activation of the hydroxylase activity. The stimulatory effect of glutamine was independent of and additive to the enhancement of N6-hydroxylation by the active component(s) in the supernatant, S2 fraction. Glutamic acid-gamma-semihydrazide activated slightly in the absence of glutamine but activation of the system by glutamine was decreased by this compound. Azaserine was shown to be an uncompetitive inhibitor with respect to lysine and this inhibition was not reversed by glutamine.

Inhibition of increased synthesis of glomerular basement membrane collagen in diabetic rats in vivo.

Diabetic rats were treated intraperitoneally for 2 week with(+)-catechin in two different dosages or calcium dobesilate and the influence of these substances on protein and basement membrane collagen synthesis was investigated. Both drugs did not alter the diabetic metabolic situation. At the lower dose, (+)-catechin led to a slight decreased and at the higher dose to a small increase of basement membrane collagen synthesis. But compared to untreated controls these differences were statistically not significant. Calcium dobesilate inhibited markedly the raised basement membrane collagen synthesis. However, this effect does not appear to be specific, since the raised general protein synthesis was also reduced. These findings show that the disturbed basement membrane metabolism in diabetes can be influenced without altering the metabolic situation.

Studies on the effect of (+)catechin on connective tissue in the guinea pig and in organ culture.

Decreased biosynthesis of (14C) hydroxyproline in skin collagen was observed after injection of the flavonoid (+)catechin into guinea pigs. No alteration in already synthesized collagens was noted, as measured by the content of hydroxyproline and hydroxylysine. No alteration in the content of skin acid glycosaminoglycans was observed. The variable binding of (+)catechin to different tissues was demonstrated. No special sensitivity to the drug of any collagen type was found.

[Pathogenesis of diabetic microangiopathy. Protein and basement membrane synthesis in isolated kidney glomeruli of diabetic and non-diabetic rats]. / Untersuchungen zur Pathogenese der diabetischen Mikroangiopathie. Protein- und Basalmembran-Synthese in isolierten Nierenglomerula diabetischer und nicht-diabetischer Ratten.

In incubation experiments with isolated glomeruli, an increased synthesis of protein and basement membranes was detected in streptozotocin-diabetic rats compared to metabolically healthy controls. Chemical analysis of isolated basement membranes and incorporation studies did not give any indication of enhanced hydroxylation of lysine in the diabetic membrane. Different glucose concentration in the incubation medium and insulin in vitro did not influence protein and basement membrane synthesis of non-diabetic glomeruli. On the other hand, in diabetic glomeruli the synthetic activity depends on glucose concentration. Insulin had a stimulatory effect on protein and basement membrane synthesis diminished at lower glucose concentration and did not inhibit the increased synthetic activity demonstrated at higher glucose concentration. Therefore, these results may be attributable to an energy deficit of incubated glomeruli and not to a lower glucose stimulation of synthesis. By treatment of diabetic rats with insulin in vivo the synthetic activity was not affected by brief normalization of blood sugar. Insulin treatment from the beginning of diabetes only lead to a normalization of protein synthesis in moderate metabolic control. On the other hand, a rise of basement membrane synthesis could only be prevented by strict metabolic control of the rats. These results show that basement membrane synthesis reacts more sensitively to the diabetic situation than overall protein synthesis. Insulin deficiency does not appear to be one of the factors directly influencing basement membrane synthesis.

Studies on the formation of N6-hydroxylysine in cell-free extracts of Aerobacter aerogenes 62-1.

We have investigated conditions optimal for the conversion of L-lysine to its N6-hydroxy derivative by partially purified cell-free extracts of Aerobacter aerogenes 62-1. The enzyme system was highly specific to L-lysine: the D-isomer and, the N2- or N6-derivatives of lysine, and alpha-amino acids were not hydroxylated. Most of the latter compounds had little effect onthe hydroxylation of L-lysine. However, -l-glutamic acid and L-glutamine enhanced the hydroxylation, with half-maximal activation achieved at 100 micrometers concentration of the effector. The Km values for pyruvate and L-(+)-lactate (compounds known to stimulate N-hydroxylysine formation) were found to be approx. 100 micrometers. The data show that N-hydroxylation of the amino acid precedes acylation in the biosynthesis of hydroxamic acid in A. aerogenes 62-1.

Effect of nonsupressible insulin-like protein on glomerular protein synthesis.

Partially purified preparations of NSILP were evaluated for effect on incorporation of labeled precursors into protein, collagen, and basement membrane by isolated rat renal glomeruli. Dowex-adsorbed NSILP (0.5 mU/mg) inhibited the secretion of protein and collagen and resulted in a significant diminution of incorporation of labeled lysine into glomerular basement membrane. Glomerular protein and collagen secretion were also inhibited in the presence of Sephadex G-200 NSILP (4.5 mU/mg), but an effect on incorporation of radiolabeled proline, hydroxyproline, lysine, and hydroxylysine into glomerular basement membrane was not observed. The in vitro response of renal glomeruli to NSILP constitutes another "insulin-like" effect of this substance.

Effect of metabolites on epsilon-N-hydroxylysine formation in cell-free extracts of Aerobacter aerogenes 62-1.

The conversion of L-lysine to its corresponding epsilon-N-hydroxy derivative has been achieved for the first time by cell-free extracts of Aerobacter aerogenes 62-1. Partial fractionation by differential centrifugation (at 12 000 X g) revealed that both supernatant and pellet are essential for maximum enzymatic activity. The omega-N-hydroxylase (EC 1.14.99) was found to function optimally at pH 7-7.5 and exhibited an apparent Km of about 75 muM for L-lysine. L(+)-Lactate or DL-lactate and pyruvate greatly stimulate the omega-N-hydroxylase activity. The system is strongly inhibited by arsenite and sulfite.

Biochemical basis of skeletal defects induced by hydralazine: inhibiton of collagen synthesis and secretion in embryonic chicken cartilage in vitro.

Hydralazine (1-hydrazinophthalazine) produces skeletal defects resembling those observed in experimentally induced manganese deficiencies. Since glycosylation of collagen, a step preceding its secretion, requires Mn2+, the effect of hydralazine on collagen secretion and the formation hydroxylysine glycosides was examined in explants of embryonic chicken long-bone rudiments. Auto-radiographic studies showed that hydralazine blocked collagen secretion. Secretion was restored by Fe2+ alone or Fe2+ + Mn2+ but not by Mn2+ alone, suggesting that a Fe2+-requiring step was involved. Biochemical analyses showed that hydralazine inhibited the formation of hydroxylysine, a step requiring Fe2+, but it did not inhibit the formation of hydroxylysine-glocosides by Mn2+-requiring steps, although the reaction was inhibited in vitro. Hydralazine also failed to inhibit intracellular mucopolysaccharide synthesis which involves several Mn2+-requiring enzymes. These observations suggest that the deleterious effects of hydralazine on bones are caused by its inhibition of hydroxylation steps in collagen synthesis.

In vitro inhibition of collagen cross links by catechol analogs.

Catechol analogs inhibit the formation of hydroxylysine-derived intermolecular collagen cross links in tissue cultures of chick embryo calvaria. Formation of intermolecular collagen cross links was measured following incorporation of [14C]lysine, reduction with sodium borohydride, and elution from an ion exchange column with a pyridine-formate gradient. Cultures grown in the presence of 10(-3) M catechol, 10(-3) M dopamine, 10(-3) M L-dopa, or 10(-3) M D,L-serine-(2,3,4-trihydroxybenzyl)-hydrazide demonstrated between 43 and 84% inhibition of hydroxylysine formation. Collagen biosynthesis was not diminished in these cultures as compared to controls without additions or with beta-aminopropionitrile when measured by collagenase digestion. The formation of hydroxylysine-derived intermolecular cross links was inhibited 34 to 93% for 5,5'-dihydroxylysinonorleucine and 7 to 71% for 5-hydroxylysinonorleucine. The catechol analogs also inhibit the activity of lysyl hydroxylase as measured by specific tritium release as triated water from an L-[4,5-3H]lysine-labeled unhydroxylated collagen substrate prepared from chick calvaria. Since catechol analogs inhibit the formation of hydroxylysine in a cell-free assay, these compounds must pass into the cells of calvaria in this culture system to inhibit intracellular hydroxylysine formation and subsequently to diminish the reducible intermolecular cross links of the newly synthesized collagen.

Effect of L-azetidine-2-carboxylic acid on glycosylations of collagen in chick-embryo tendon cells.

The glycosylations of hydroxylysine during collagen biosynthesis in isolated chick-embryo tendon cells were studied by using pulse-chase labelling experiments with [14C]-lysine. The hydroxylation of lysine and the glycosylations of hydroxylysine continued after a 5 min pulse label for up to about 10 min during the chase period. These data differ from those obtained previously in isolated chick-embryo cartilage cells, in which, after a similar 5 min pulse label, these reactions continued during the chase period for up to about 20 min. The collagen synthesized by the isolated chick-embryo tendon cells differed markedly from the type I collagen of adult tissues in its degree of hydroxylation of lysine residues and glycosylations of hydroxylysine residues. When the isolated tendon cells were incubated in the presence of L-azetidine-2-carboxylic acid, the degree of glycosylations of hydroxylysine during the first 10 min of the chase period was identical with that in cells incubated without thcarboxylic acid for at least 60 min, whereas no additional glycosylations took place in the control cells after the 10 min time-point. As a consequence, the collagen synthesized in the presence of this compound contained more carbohydrate than did the collagen synthesized by the control cells. Additional experiments indicated that azetidine-2-carboxylic acid did not increase the collagen glycosyltransferase activities in the tendon cells or the rate of glycosylation reactions when added directly to the enzyme incubation mixture. Control experiments with colchicine indicated that the delay in the rate of collagen secretion, which was observed in the presence of azetidine-2-carboxylic acid, did not in itself affect the degree of glycosylations of collagen. The results thus suggest that the increased glycosylations were due to inhibition of the collagen triple-helix formation, which is known to occur in the presence of azetidine-2-carboxylic acid.

Cortisol effect on collagen biosynthesis in embryonic explants and in vitro hydroxylation of protocollagen.

The effect of increasing doses of hydrocortisone acetate, hydrocortisone phosphoric acid complex, and hydrocortisone sodium succinate on collagen biosynthesis was assayed in two different systems. I. Explants of chicken embryo tibiae showed decreased biosynthesis of [14C]hydroxyproline and total and glycosylated [14C]hydroxylysine under the influence of high doses of hydrocortisone. With the lower doses of hydrocortisone acetate, no effect was noticed. The total uptake of the precursor amino acids followed patterns similar to those of collagen biosynthesis. II. Hydroxylation of [14C]proline labelled protocollagen by protocollagen proline hydroxylase was inhibited by high doses of hydrocortisone acetate, hydrocortisone phosphoric acid complex, and hydrocortisone sodium succinate. III. A decreased diffusion of collagen to the medium with increasing doses of hydrocortisone acetate, hydrocortisone phosphoric acid, and hydrocortisone sodium succinate was noticed. IV. No further hydroxylation of new-synthesized collagen was obtained under the influence of hydrocortisone phosphoric acid and hydrocortisone sodium succinate, when the undialyzable material was used as a substrate for protocollagen proline hydroxylase.

Stimulation of collagen galactosyltransferase and glucosyltransferase activities by lysophosphatidylcholine.

Lysophosphatidylcholine stimulated the activities of collagen galactosyl- and glucosyl-transferases in chick-embryo extract and its particulate fractions in vitro, whereas essentially no stimulation was noted in the high-speed supernatant, where the enzymes are soluble and membrane-free. The stimulatory effect of lysophosphatidylcholine was masked by 0.1% Triton X-100. In kinetic experiments lysophosphatidylcholine raised the maximum velocities with respect to the substrates and co-substrates, whereas no changes were observed in the apparant Km values. Phospholipase A preincubation of the chick-embryo extract resulted in stimulation of both transferase activities, probably gy generating lysophosphatides from endogenous phospholipids. No stimulation by lysophosphatidylcholine was found when tested with 500-fold-purified glycosyltransferase. The results suggest that collagen glycosyltransferases must be associated with the membrane structures of the cell in order to be stimulated by lysophosphatidylcholine. Lysophosphatidylcholine could have some regulatory significance in vivo, since its concentration in the cell is comparable with that which produced marked stimulation in vitro.

Defects in the biochemistry of collagen in diseases of connective tissue.

The collagens are the major structural glycoproteins of connective tissues. A unique primary structure and a multiplicity of post-translational modification reactions are required for normal fibrillogenesis. The post-translational modifications include hydroxylation of prolyl and lysyl residues, glycosylation, folding of the molecule into triple-helical conformation, proteolytic conversion of precursor procollagen to collagen, and oxidative deamination of certain lysyl and hydroxylysyl residues. Any defect in the normal mechanisms responsible for the synthesis and secretion of collagen molecules or the deposition of these molecules into extracellular fibers could result in abnormal fibrillogenesis; such defects could result in a connective tissue disease. Recently, defects in the regulation of the types of collagen synthesized and in the enzymes involved in the post-translational modifications have been found in heritable diseases of connective tissue. Thus far, the primary heritable disorders of collagen metabolism in man include lysyl hydroxylase deficiency in Ehlers-Danlos syndrome type VI, p-collagen peptidase deficency in Ehlers-Danlos syndrome type VII, decreased synthesis of type III collagen in Ehlers-Danlos syndrome type IV, lysyl oxidase deficency in S-linked cutis laxa and Ehlers-Danlos syndrome type V, and decreased synthesis of type I collagen in osteogenesis imperfecta.

Human glomerular smooth muscle (mesangial) cells in culture.

Glomerular smooth muscle cells were grown and subcultured from human glomeruli in vitro. These cells, stained with antiserum to smooth muscle actomyosin and examined by immunofluorescence, had brightly stained intracellular fibrils similar to those seen in smooth muscle cells. Actomyosin fibrils were altered by conditions affecting actomyosin in vitro. Glomerular smooth muscle cells lacked the antihemophilic factor and blood group antigens present in endothelial cells, and are, therefore, most likely derived from the smooth muscle cells of the glomerular mesangium. By radio-labeled amino acid analysis, they synthesize a collagen differing from that of fibroblasts, and which probably differs from basement membrane collagen. Other cell types could be grown and subcultured using a different glomerular isolation technique, culture medium and method of subculture.

The effect of diabetes on renal lysine utilization.

Current evidence indicates that a hydroxylysine-rich glycoprotein may be of importance in the structural organization and accumulation of glomerular basement membrane in the diabetic state. To further evaluate the role of insulin deficiency in renal glycoprotein synthesis, the effect of experimental diabetes on the incorporation and hydroxylation of 14C-lysine by cell-free systems prepared from rat renal cortex was examined. Microsomal protein synthesis was increased in diabetic preparations, but the rise in renal cortical collagen synthesis relative to total protein synthesis was greater. These changes were not duplicated by the addition of a mixture of unlabeled amino acids or hydroxylation cofactors to incubations with preparations from normal animals.