Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress.
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BACKGROUND: The tissue accumulation of protein-bound advanced glycation endproducts (AGE) may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS) and nitric oxide synthase (NOS) expression on these AGE-collagen mediated effects. RESULTS: AGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP) activity. In preosteoblastic MC3T3E1 cells (24-hour culture), proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts) AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture) AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines. CONCLUSIONS: These results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways. (+info)
Temporal and spatial mRNA expression of bone sialoprotein and type I collagen during rodent tooth movement.
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To investigate the mechanism of bone formation during tooth movement, in situ hybridization was performed with digoxigenin-labelled RNA probes to detect bone sialoprotein (BSP) and type I collagen mRNAs in the dentoalveolar tissue of 72 Sprague-Dawley rats. An elastic band was inserted between the first and second right maxillary molars, and the teeth experimentally moved for 1, 3, and 7 days. The left first maxillary molar was used as the control. For the untreated molars, osteoblasts and osteocytes near the distal surface of the interradicular septum (IRS) expressed a high level of both BSP and type I collagen mRNAs, while cells on the mesial side of the IRS showed a low level of these mRNAs. For the first molars subjected to experimental tooth movement, a high level of type I collagen mRNA expression was found in the osteoblasts on the tension side of the IRS after 1 day of experimental tooth movement. A high level of BSP mRNA was detected after 3 days of experimental tooth movement. However, a negligible amount of both mRNAs was found in cells on the compression side. These results support the hypothesis that BSP may be involved in mineralization during physiological bone remodelling. On application of orthodontic force, osteoblasts were activated and induced to express BSP mRNA, which is involved in bone remodelling due to orthodontic force. In addition, response to the orthodontic force was observed in osteocytes. (+info)
Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of hydroxyproline in collagen folding and fibril formation.
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Human unhydroxylated homotrimeric triple-helical collagen I produced in transgenic plants was used as an experimental model to provide insights into the role of hydroxyproline in molecular folding and fibril formation. By using chemically cross-linked molecules, we show here that the absence of hydroxyproline residues does not prevent correct folding of the recombinant collagen although it markedly slows down the propagation rate compared with bovine fully hydroxylated homotrimeric collagen I. Relatively slow cis-trans-isomerization in the absence of hydroxyproline likely represents the rate-limiting factor in the propagation of the unhydroxylated collagen helix. Because of the lack of hydroxylation, recombinant collagen molecules showed increased flexibility as well as a reduced melting temperature compared with native homotrimers and heterotrimers, whereas the distribution of charged amino acids was unchanged. However, unlike with bovine collagen I, the recombinant collagen did not self-assemble into banded fibrils in physiological ionic strength buffer at 20 degrees C. Striated fibrils were only obtained with low ionic strength buffer. We propose that, under physiological ionic strength conditions, the hydroxyl groups in the native molecule retain water more efficiently thus favoring correct fibril formation. The importance of hydroxyproline in collagen self-assembly suggested by others from the crystal structures of collagen model peptides is thus confirmed experimentally on the entire collagen molecule. (+info)
A 36-amino-acid region of CIITA is an effective inhibitor of CBP: novel mechanism of gamma interferon-mediated suppression of collagen alpha(2)(I) and other promoters.
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The class II transactivator (CIITA) is induced by gamma interferon (IFN-gamma) and activates major histocompatibility complex class II; however, this report shows it suppresses other genes. An N-terminal 36 amino acids of CIITA mediates suppression of the collagen alpha(2)(I) promoter via binding to CREB-binding protein (CBP). Reconstitution of cells with CBP reverts this suppression. IFN-gamma is known to inhibit collagen gene expression; to test if CIITA mediates this gene suppression, a mutant cell line defective in CIITA induction but not in the activation of STAT1/JAK/IRF-1 is studied. IFN-gamma suppression of the collagen promoter and the endogenous gene is observed in the wild-type control but not in the mutant line. Suppression is restored when CIITA is introduced. Other targets of CIITA-mediated promoter suppression include interleukin 4, thymidine kinase, and cyclin D1. (+info)
Lefty contributes to the remodeling of extracellular matrix by inhibition of connective tissue growth factor and collagen mRNA expression and increased proteolytic activity in a fibrosarcoma model.
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Homeostasis of the extracellular matrix (ECM) of tissues is regulated by controlling deposition and degradation of ECM proteins. The breakdown of ECM is essential in blastocyst implantation and embryonic development, tissue morphogenesis, menstrual shedding, bone formation, tissue resorption after delivery, and tumor growth and invasion. TGF-beta family members are one of the classes of proteins that actively participate in the homeostasis of ECM. Here, we report on the effect of lefty, a novel member of the TGF-beta family, on the homeostasis of extracellular matrix in a fibrosarcoma model. Fibroblastic cells forced to express lefty by retroviral transduction lost their ability to deposit collagen in vivo. This event was associated with down-regulation of the steady-state level of connective tissue growth factor that induces collagen type I mRNA. In addition, lefty transduction significantly decreased collagen type I mRNA expression and simultaneously increased collagenolytic, gelatinolytic, elastolytic, and caseinolytic activities in vivo by the transduced fibroblasts. These findings provide a new insight on the actions of lefty and suggest that this cytokine plays an active role in remodeling of the extracellular matrix in vivo. (+info)
Dipyridamole inhibits TGF-beta-induced collagen gene expression in human peritoneal mesothelial cells.
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BACKGROUND: Peritoneal matrix accumulation is characteristic of peritoneal fibrosis (PF). Continuous ambulatory peritoneal dialysis (CAPD) patients who had persistent transforming growth factor-beta (TGF-beta) in their drained effluent had an increased risk of PF. We previously reported that TGF-beta stimulates the expression of types I and III collagen mRNA in cultured human peritoneal mesangial cells (HPMCs), which may predispose them to develop PF. Pharmacological interventions to attenuate TGF-beta-stimulated matrix accumulation in HPMC may have therapeutic potential for the treatment of PF. The SMAD family and the extracellular signal-regulated protein kinase (ERK1/2, p44/p42) pathways have been shown to participate in TGF-beta signaling. Our current study identified these signal pathways in HPMCs and investigated the molecular mechanisms involved in the inhibitory effects of dipyridamole on TGF-beta-induced collagen gene expression in HPMCs. METHODS: HPMCs were cultured from human omentum by an enzyme digestion METHOD: Expression of collagen alpha1(I) mRNA was determined by Northern blotting. The SMAD proteins and the ERK1/2 activity were determined by Western blotting. RESULTS: TGF-beta-stimulated collagen alpha1(I) mRNA expression of HPMC was inhibited by dipyridamole in a dose-dependent manner. Smad2 and ERK1/2 were activated in response to TGF-beta; however, TGF-beta had little effect on the protein expression of Smad4. The addition of PD98059, which blocked activation of ERK1/2, suppressed TGF-beta-induced collagen alpha1(I) mRNA expression in a dose-dependent manner. At a concentration that inhibited collagen gene expression (17 microg/mL), dipyridamole suppressed ERK1/2 activation by TGF-beta. In contrast, the same concentration of dipyridamole had no effect on TGF-beta-induced activation of Smad2. CONCLUSION: Dipyridamole inhibits TGF-beta-induced collagen gene expression in HPMC through modulation of the ERK pathway. Our study of dipyridamole may provide therapeutic basis for clinical applications in the prevention of PF. (+info)
Transforming growth factor beta1 (TGF-beta1) promotes endothelial cell survival during in vitro angiogenesis via an autocrine mechanism implicating TGF-alpha signaling.
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Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Transforming growth factor beta1 (TGF-beta1) alone can substitute for serum and induce cell survival and tubular network formation. This TGF-beta1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-beta1-induced angiogenesis mainly by compromising cell survival. We established that TGF-beta1 stimulated the expression of TGF-alpha mRNA and protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all these TGF-beta1-mediated signaling events, including tubular network formation, were suppressed by incubating TGF-beta1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-alpha alone poorly stimulated angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-beta1. We therefore propose that TGF-beta1 promotes angiogenesis at least in part via the autocrine secretion of TGF-alpha, a cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK. (+info)
Urinary cross-linked N-telopeptides of type I collagen and bone metabolic diseases.
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OBJECTIVE: To evaluate the type I collagen cross-linked N-telopeptide (NTx) levels in human urine as an indicator of bone resorption rate in different ages, sex and in bone metabolic diseases. METHODS: Urinary NTx was determined by immunoassay in 591 Beijing healthy subjects aged from 0 to 86 years and 379 patients with bone metabolic diseases. RESULTS: The levels of urinary NTx were significantly higher in children than in adults (P < 0.001) and higher in boys than in girls (P < 0.01) and increased 1.4-2.2 times in postmenopausal females than in men and premenopausal women. Urine NTx had a positive linear correlation with urine HOP/Cr (r = 0.778, P < 0.01) and Ca/Cr ratio (r = 0.320, P < 0.01), and a negative linear correlation with age (r = -0.523, P < 0.01) and lumbar spine BMD (r = -0.426, P < 0.01). The levels of urine NTx increased for 3.6 times in pregnancy, 1.5 times in osteoporosis, 1.9 times in fragility fracture, 3.6 times in chronic renal failure, 2.1 times in rickets and 7.2 times in multiple myeloma compared to age matched controls. CONCLUSIONS: NTx in urine is a specific and sensitive indicator of bone resorption and is able to distinguish normal premenopause from late osteoporotic patients. NTx could be used as diagnostic information about metabolic bone diseases, and to monitor antiresorptive therapy. (+info)