Extracellular-signal regulated kinase signaling pathway mediates downregulation of type I procollagen gene expression by FGF-2, PDGF-BB, and okadaic acid in osteoblastic cells

Although basic fibroblast growth factor (FGF-2) had been shown to inhibit type I collagen gene expression in osteoblast, its inhibitory mechanism is unknown. In the present study, we investigated the underlying mechanisms by which growth factors downregulate type I collagen gene expression. Treatment of mouse osteoblastic MC3T3-E1 cells with okadaic acid (40 ng/ml), an inhibitor of phosphoserine/threonine-specific protein phosphatase and activator of ERK1/2, for 24 h and 48 h completely inhibited steady-state mRNA levels of type I collagen. FGF-2 (30 ng/ml), platelet-derived growth factor-BB (PDGF-BB), 30 ng/ml, and serum, which activate ERK mitogen-activated protein kinase (MAPK) pathway also inhibited collagen type I gene expression, suggesting that the activation of ERK pathway mediates inhibition of type I collagen mRNA. This observation was further confirmed by experiments using inhibitors of the ERK pathway (i.e., PD and U0126), which increased type I collagen mRNA in MC3T3-E1 cells, indicating that the inhibition of ERK pathway upregulates type I collagen gene expression. Low serum (0.3%) markedly increased type I collagen mRNA. MEK inhibitor PD inhibited c-fos induction by FGF-2 and PDGF-BB, suggesting that c-fos is the downstream target of ERK pathway. Our data have clearly demonstrated for the first time that the ERK MAPK pathway play an important role in the regulation of type I collagen gene expression in osteoblastic cells. Results also showed that one of the mechanisms by which FGF-2 and PDGF-BB downregulate type I collagen gene expression in the osteoblast is through the activation of ERK signaling pathway.     

Notch3对促进胰腺星形细胞活化的基因表达及信号通路的影响

目的:分离培养小鼠胰腺星形细胞(PSCs),检测Notch3对促进PSCs活化的基因表达及信号通路的影响.方法:对小鼠PSCs进行分离培养及传代.采用免疫荧光染色检测活化的小鼠PSCs中α-SMA,fibronectin及collagen Ⅰ的表达;细胞分组为空白对照组(MOCK组),阴性对照组(转染Notch3 si...     

Effects of tripterine on mRNA expression of TGF-beta1 and collagen IV expression in BW F1 mice

Tripterine is a chemical isolated from a traditional Chinese herb which had been testified for its anti-inflammatory and immunosuppressive activities in a previous study. However, little is known about the effects and mechanism of action of Tripterine on treating lupus nephritis. In the present study we investigated the effect of Tripterine on the F1 hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice which functioned as a model of human systemic lupus erythematosus (BW F1 mice) and evaluated the possible mechanism implicated in the mRNA expression of TGF-beta1 and collagen IV expression of the BW F1 mice kidney tissue. Different doses of Tripterine were injected peritoneally to BW F1 mice at different stages to study the preventive effects of Tripterine on lupus nephritis glomerulosclerosis and its mechanisms. Twenty-four hour urine protein excretion, serum anti-dsDNA antibodies and the expression of collagen type IV were examined by immunohistochemistry while the expression of TGF-beta1 mRNA was detected by RT nested PCR. Tripterine decreased urine protein excretion and the level of serum anti-dsDNA antibodies and also suppressed the expression of collagen type IV and TGF-beta1 mRNA in the murine kidney tissue. Administration of Tripterine before the occurrence of proteinuria had much greater protective effects than if it was administered after the occurrence of proteinuria. No significant difference was found between the 3 mg/kg/week Tripterine-treated-group and the 6 mg/kg/week Tripterine-treated-group. Tripterine had a definite protective effect on glomerulosclerosis of the lupus murine model. Tripterine could significantly reduce the amount of urine protein excretion, suppress the formation of serum anti-dsDNA antibodies, it could also efficiently decrease the expression of renal collagen type IV probably due to its suppressive effect on the expressions of local TGF-(1 mRNA) in this model.     

Advanced glycation end-product-induced mitogenesis and collagen production are dependent on angiotensin II and connective tissue growth factor in NRK-49F cells

Diabetic nephropathy (DN) is characterized by glomerulopathy and tubulointerstitial expansion followed by renal fibrosis. Angiotensin II (Ang II) and connective tissue growth factor (CTGF) are involved in the pathogenesis of DN, while Janus kinase 2 (JAK2) is important in advanced glycation end-product (AGE)-induced effects in renal interstitial (NRK-49F) fibroblasts. Thus, we studied the role of Ang II, CTGF, and JAK2 in AGE-induced effects in NRK-49F cells. We found that AGE (150 microg/ml) increased mitogenesis and type I collagen production at 7 days while Ang II (10(-7)M) increased mitogenesis and type I collagen production at 3 days. We also found that AGE (150 microg/ml) increased angiotensinogen protein at 2 days, which was attenuated by AG-490 (a JAK2 inhibitor). AGE (150 microg/ml) increased CTGF mRNA and protein expression at 3 and 5 days, respectively. Ang II (10(-7)M) increased CTGF mRNA and protein expression at 1 and 2 days, respectively, which were attenuated by AG-490. Moreover, losartan (a type I angiotensin receptor blocker) and captopril (an angiotensin converting enzyme inhibitor) attenuated AGE-induced CTGF mRNA/protein expression while attenuating AGE-induced mitogenesis and type I collagen production. AG-490 and CTGF antisense (but not sense) oligodeoxynucleotide (ODN) attenuated Ang II (10(-7)M) and AGE-induced mitogenesis and type I collagen production at 3 and 7 days, respectively. We concluded that AGE (150 microg/ml)-induced mitogenesis and type I collagen production are dependent on the Ang II-JAK2-CTGF pathway in NRK-49F cells. Moreover, Ang II-induced mitogenesis and type I collagen production are dependent on the JAK2-CTGF pathway.     

Substitution of murine type I collagen A1 3-hydroxylation site alters matrix structure but does not recapitulate osteogenesis imperfecta bone dysplasia

Null mutations in CRTAP or P3H1, encoding cartilage-associated protein and prolyl 3-hydroxylase 1, cause the severe bone dysplasias, types VII and VIII osteogenesis imperfecta. Lack of either protein prevents formation of the ER prolyl 3-hydroxylation complex, which catalyzes 3Hyp modification of types I and II collagen and also acts as a collagen chaperone. To clarify the role of the A1 3Hyp substrate site in recessive bone dysplasia, we generated knock-in mice with an α1(I)P986A substitution that cannot be 3-hydroxylated. Mutant mice have normal survival, growth, femoral breaking strength and mean bone mineralization. However, the bone collagen HP/LP crosslink ratio is nearly doubled in mutant mice, while collagen fibril diameter and bone yield energy are decreased. Thus, 3-hydroxylation of the A1 site α1(I)P986 affects collagen crosslinking and structural organization, but its absence does not directly cause recessive bone dysplasia. Our study suggests that the functions of the modification complex as a collagen chaperone are thus distinct from its role as prolyl 3-hydroxylase.     

The effect of transforming growth factor-beta on cell proliferation and collagen formation by lung fibroblasts

We examined the effects of transforming growth factor-beta (TGF-beta) on the production of collagen by cultures of human embryonic lung fibroblasts. TGF-beta at 0.1 ng/ml appeared to activate selectively extracellular collagen accumulation as compared with total protein production. A maximal effect inducing a 2-3-fold increase in collagen and total protein production occurred at a dose of 1.0 ng/ml in fibroblast cultures. TGF-beta had no effect on fibroblast proliferation after a 24- and 48-h exposure, including cultures that received a second dose after 24 h. Collagenase digestion of radiolabeled collagen derived from TGF-beta-treated and -untreated cultures revealed no differences in the extent of hydroxylation (37.3 versus 33.4%). TGF-beta increased the production of types I and III collagen without affecting the proportion of collagen types. Fibroblast cultures maintained in medium containing TGF-beta sustained an activated rate of collagen production of 5 nmol/ml/24 h over at least 72 h. We found that epidermal growth factor slightly enhanced TGF-beta-induced collagen formation, whereas TGF-beta increased the proliferative effect of epidermal growth factor. Taken together, these data indicate that collagen production and cell proliferation can be independently regulated and that TGF-beta may have a role in the resolution of tissue injury by stimulating fibroblast-derived collagen synthesis.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5–1 μg/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 μg/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 μg/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 μg/ml). Somatostatin (1 μg/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated.The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

Features of formation of phases of dual-phase polymeric system in the presence of collagen I, laminin I, and their mixtures

A study was made of the phase formation kinetics of a two-phase aqueous polymer system, consisting of 7.8% (w/w) dextran-500 and 4.6% (w/w) polyethylene glycol-6000, in the presence of various concentrations of collagen (0.07-0.20 mg/ml), laminin I (0.01-0.03 mg/ml) and their mixture. The phase formation was evaluated by registration of its optical density on a spectrophotometer. The obtained two-phase polymer system optical density curves and also the partitioning coefficients for the studied objects depend on surface properties of these objects. It has been shown that the surface properties of collagen I and laminin I differ according to differences in their molecules conformations, and that the phase formation kinetics points to their interaction during a mutual partitioning of these proteins in the system. The authors made a conclusion that collagen I and laminin I in the two-phase polymer system conditions could make complexes.     

Mice lacking serum amyloid P component do not necessarily develop severe autoimmune disease

Interleukin-10 (IL-10) is a cytokine with many regulatory functions. In particular, IL-10 exerts neutralizing effect on other cytokines, and therefore IL-10 is thought to have important therapeutic implications. Recent reports suggest that IL-10 regulates not only immunocytes but also collagen and collagenase gene expression in fibroblasts. In this study, we investigated the effect of IL-10 on gene expression of extracellular matrix (ECM) proteins, such as type I collagen, fibronectin, and decorin, in human skin fibroblasts. Results of Northern blot analysis showed that both collagen I and fibronectin mRNAs were downregulated, while decorin gene expression was enhanced by IL-10 (10 ng/ml) time-dependently (6-24 h). alpha1(I) collagen and fibronectin mRNAs were decreased to one-third and one-fourth, respectively, by 50 ng/ml IL-10, whereas decorin mRNA was increased up to 2.7-fold by 50 ng/ml IL-10. Response to IL-10 by scleroderma fibroblasts was similar to that in normal dermal fibroblasts, with decreased expression levels of collagen and fibronectin and induced decorin mRNA levels. Transforming growth factor-beta (TGF-beta) is a crucial fibrogenic cytokine which upregulates the mRNA expression of collagen and fibronectin, whereas it downregulates decorin mRNA expression in fibroblasts. Monocyte chemoattractant protein-1 (MCP-1) has recently been shown to upregulate the type I collagen mRNA expression in cultured fibroblasts. We therefore examined whether IL-10 alters gene expression of ECM elicited by TGF-beta and MCP-1. Our results demonstrated that IL-10 downregulated the TGF-beta-elicited increase of mRNA expression of type I collagen and fibronectin, while partially recovering TGF-beta-elicited decrease of decorin expression in normal skin fibroblasts. By contrast, IL-10 did not alter the MCP-1-elicited upregulation of mRNA expression of either alpha1(I) collagen and decorin. Our data indicate that IL-10 differentially regulates TGF-beta and MCP-1 in the modulation of ECM proteins and therefore suggest that IL-10 plays a role in the regulation of tissue remodeling.     

Secretion of biologically active human granulocyte colony-stimulating factor (G-CSF) in milk of transgenic mice

Two constructs were devised, containing the full-length gene of the human granulocyte colony-stimulating factor (G-CSF) fused with the 5' and 3' flanking promoter sequences of bovine alpha-S1-casein gene. Both constructs contained a 1518-bp fragment that included exons 18 and 19 and 320 bp of the 3' flanking region of bovine gene @CSN1S1, but differed in size of the 5' flanking sequences, which were of 721 bp, and exon 1 in construct pGCm1 and 2001 bp and exon 1 and intron 1 in construct pGCm2. With both constructs, transgenic mice were produced. The transgene expression was assessed using RT-PCR and immunochemically from the production of human G-CSF in milk of lactating females. Secretion of human G-CSF into the milk varied in a wide range, from 0.8 microg/ml to over 1 mg/ml, in mice with construct pGCml and was low (up to 60 microg/ml) or absent in mice with construct pGCm2. G-CSF glycosylation was incomplete in mice with transgene pGCml and complete in mice with pGCm2. G-CSF of transgenic mouse milk was shown to stimulate the formation and growth of granulocyte-containing colonies in human umbilical blood cell culture and be close or identical in physiological activity to the natural human G-CSF.