Procollagen binds to both prolyl 4-hydroxylase/protein disulfide isomerase and HSP47 within the endoplasmic reticulum in the absence of ascorbate.
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In cells, only properly folded procollagen trimers are secreted from the endoplasmic reticulum (ER), while improperly folded abnormal procollagens are retained within the ER. Ascorbic acid is a co-factor in procollagen hydroxylation, which in turn is required for trimer formation. We examined chaperone proteins which bound to procollagen in the absence of ascorbic acid, a model which mimics the human disease scurvy at the cellular level. We found that both prolyl 4-hydroxylase (P4-H)/protein disulfide isomerase (PDI) and HSP47 bound to procollagen in the absence of ascorbic acid. However, the binding of PDI to procollagen decreased when HSP47 was co-transfected, suggesting that HSP47 and PDI compete for binding to procollagen. These data indicate that P4-H/PDI and HSP47 have cooperative but distinct chaperone functions during procollagen biosynthesis. (+info)
Insulin-like growth factor system components in hyperparathyroidism and renal osteodystrophy.
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BACKGROUND: The insulin-like growth factor (IGF) system plays a key role in regulation of bone formation. In patients with renal osteodystrophy, an elevation of some IGF binding proteins (IGFBPs) has been described, but there is no study measuring serum levels of both IGF-I and IGF-II as well as IGFBP-1 to -6 in different forms of renal osteodystrophy and hyperparathyroidism. METHODS: In a cross-sectional study, we investigated 319 patients with mild (N = 29), moderate (N = 48), preuremic (N = 37), and end-stage renal failure (ESRF; N = 205). The ESRF group was treated by hemodialysis (HD; N = 148), peritoneal dialysis (PD; N = 27), or renal transplantation (RTX; N = 30). As controls without renal failure, we recruited age-matched healthy subjects (N = 87) and patients with primary hyperparathyroidism (pHPT; N = 25). Serum levels of total and free IGF-I, IGF-II, IGFBP-1 to -6, and biochemical bone markers including intact parathyroid hormone (PTH), bone alkaline phosphatase (B-ALP), and osteocalcin (OSC) were measured by specific immunometric assays. IGF system components and bone markers were correlated with clinical and bone histologic findings. Mean values +/- SEM are given. RESULTS: With declining renal function a significant increase was measured for IGFBP-1 (range 7- to 14-fold), IGFBP-2 (3- to 8-fold), IGFBP-3 (1.5- to 3-fold), IGFBP-4 (3- to 19-fold), and IGFBP-6 (8- to 25-fold), whereas IGFBP-5 levels tended to decrease (1.3- to 1. 6-fold). In contrast, serum levels of IGF-I, free IGF-I, and IGF-II remained constant in most patients. Compared with renal failure patients, pHPT patients showed a similar decline in IGFBP-5 levels and less elevated levels of IGFBP-1 (3.5-fold), IGFBP-2 (2-fold), IGFBP-3 (1.2-fold), and IGFBP-6 (4-fold) but no elevation of IGFBP-4 levels. In all subjects, free and total IGF-I levels showed significant negative correlations with IGFBP-1, IGFBP-2, and IGFBP-4 (that is, inhibitory IGF system components) and significant positive correlations with IGFBP-3 and IGFBP-5 (that is, stimulatory IGF system components). A positive correlation was observed between IGF-II and IGFBP-6. ESRF patients with mixed uremic bone disease and histologic evidence for osteopenia revealed significantly (P < 0.05) higher levels of IGFBP-2 and IGFBP-4 but lower IGFBP-5 levels. Histologic parameters of bone formation showed significant positive correlations with serum levels of IGF-I, IGF-II, and IGFBP-5. In contrast, IGFBP-2 and IGFBP-4 correlated positively with indices of bone loss. Moreover, dialysis patients with low bone turnover (N = 24) showed significantly (P < 0.05) lower levels of IGFBP-5, PTH, B-ALP, and OSC than patients with high bone turnover. CONCLUSION: Patients with primary and secondary hyperparathyroidism showed lower levels of the putative stimulatory IGFBP-5 but higher levels of IGFBP-1, -2, -3, and -6, whereas total IGF-I and IGF-II levels were not or only moderately increased. The marked increase in serum levels of IGFBP-4 appeared to be characteristic for chronic renal failure. IGFBP-5 correlated with biochemical markers and histologic indices of bone formation in renal osteodystrophy patients and was not influenced by renal function. Therefore, IGFBP-5 may gain significance as a serological marker for osteopenia and low bone turnover in long-term dialysis patients. (+info)
Platelet-activating factor induces an imbalance between matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 expression in human uterine cervical fibroblasts.
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Platelet-activating factor (PAF) is involved in such reproductive processes as parturition. We investigated the effect of PAF on the expression of matrix metalloproteinase-1 (MMP-1) and that of tissue inhibitor of metalloproteinases-1 (TIMP-1) in human uterine cervical fibroblasts. Uterine cervical tissue was obtained from patients who underwent cesarean section at term. Collagenase-dispersed fibroblasts were cultured and used in the experiments. PAF receptor was identified in the uterine cervical fibroblasts by use of reverse transcription-polymerase chain reaction and Southern blot analysis. Northern blot analysis showed that PAF increased the expression of MMP-1 mRNA in a time-dependent manner, whereas expression of TIMP-1 mRNA was not affected by PAF. Concentration of MMP-1 protein in the PAF-treated culture media significantly exceeded that in control cultures. The PAF-induced production of MMP-1 protein was abolished by treatment with WEB 2170, a specific PAF receptor antagonist. Results suggest that PAF may accelerate collagenolysis in the human uterine cervix by inducing an imbalance in the activity between MMP-1 and TIMP-1, thus contributing to the cervical ripening during parturition. (+info)
Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.
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Damage to human skin due to ultraviolet light from the sun (photoaging) and damage occurring as a consequence of the passage of time (chronologic or natural aging) are considered to be distinct entities. Photoaging is caused in part by damage to skin connective tissue by increased elaboration of collagen-degrading matrix metalloproteinases, and by reduced collagen synthesis. As matrix metalloproteinase levels are known to rise in fibroblasts as a function of age, and as oxidant stress is believed to underlie changes associated with both photoaging and natural aging, we determined whether natural skin aging, like photoaging, gives rise to increased matrix metalloproteinases and reduced collagen synthesis. In addition, we determined whether topical vitamin A (retinol) could stimulate new collagen deposition in sun-protected aged skin, as it does in photoaged skin. Sun-protected skin samples were obtained from 72 individuals in four age groups: 18-29 y, 30-59 y, 60-79 y, and 80+ y. Histologic and cellular markers of connective tissue abnormalities were significantly elevated in the 60-79 y and 80+ y groups, compared with the two younger age groups. Increased matrix metalloproteinase levels and decreased collagen synthesis/expression were associated with this connective tissue damage. In a separate group of 53 individuals (80+ y of age), topical application of 1% vitamin A for 7 d increased fibroblast growth and collagen synthesis, and concomitantly reduced the levels of matrix-degrading matrix metalloproteinases. Our findings indicate that naturally aged, sun-protected skin and photoaged skin share important molecular features including connective tissue damage, elevated matrix metalloproteinase levels, and reduced collagen production. In addition, vitamin A treatment reduces matrix metalloproteinase expression and stimulates collagen synthesis in naturally aged, sun-protected skin, as it does in photoaged skin. (+info)
Collagen fibrillogenesis: intermediate aggregates and suprafibrillar order.
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Polymerization of collagen in vitro has been studied with the electron microscope at early time points of fibril assembly. We have found morphologically distinct stages of aggregation, which we suggest represent successive steps in fibril formation. Linear growth of the fibril appears to occur by the tandem addition of aggregates to each other and subsequently to the ends of a subfibril; lateral growth occurs by the entwining, like a rope, of these subfibrils. Fibrillogenesis is also accompanied by extensive development of suprafibrillar order in which various patterns of parallel, spiral, and orthogonal sets of fibrils were frequently observed. (+info)
Simultaneous synthesis of types I and III collagen by fibroblasts in culture.
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Specific antibodies against types I and III collagens and procollagens were used to localize these proteins in cultured human cells. These studies indicate that the same cell makes both proteins. No type III procollagen synthesis was observed in cells from two patients with two patients with the Ehlers-Danlos type IV syndrome. (+info)
Segment-long-spacing aggregates and isolation of COOH-terminal peptides from type I procollagen.
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Type I procollagen secreted by matrix-free cells from chick embryo tendons was purified by DEAE-cellulose chromatography. Electron microscopy of segment-long-spacing aggregates of the procollagen demonstrated the presence of both NH2-terminal and COOH-terminal extensions not found in collagen. The procollagen was digested with bacterial collagenase and the COOH-terminal fragments were isolated by gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Analysis of tryptic peptides demonstrated that the COOH-terminal extensions on the pro alpha 1 and pro alpha 2 chains had different primary structures. (+info)
Triple helix assembly and processing of human collagen produced in transgenic tobacco plants.
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The use of tobacco plants as a novel expression system for the production of human homotrimeric collagen I is presented in this report. Constructs were engineered from cDNA encoding the human proalpha1(I) chain to generate transgenic tobacco plants expressing collagen I. The recombinant proalpha1(I) chains were expressed as disulfide-bonded trimers and were shown to fold into a stable homotrimeric triple helix. Moreover, the recombinant procollagen was subsequently processed to collagen as it occurs in animals. Large amounts of recombinant collagen were purified from field grown plant material. The data suggest that plants are a valuable alternative for the recombinant production of collagen for various medical and scientific purposes. (+info)