Collagen Promoter Research Articles

BMP signaling stimulates cellular differentiation at multiple steps during cartilage development

Bone morphogenetic proteins (BMPs) play important roles at multiple stages of endochondral bone formation. However, the roles of BMP signaling in chondrocytes in vivo are still contentious. In the present study, we overexpressed a constitutively active BMP receptor 1A (caBmpr1a) in chondrocytes by using two systems: caBmpr1a was directly driven by a rat type II collagen promoter in a conventional transgenic system and indirectly driven in a UAS-Gal4 binary system. CaBmpr1a expression caused shortening of the columnar layer of proliferating chondrocytes and up-regulation of maturation markers, suggesting acceleration of differentiation of proliferating chondrocytes toward hypertrophic chondrocytes. In addition to the acceleration of chondrocyte differentiation, conventional transgenic mice showed widening of cartilage elements and morphological alteration of perichondrial cells, possibly due to stimulation of differentiation of prechondrogenic cells. Moreover, bigenic expression of caBmpr1a rescued the differentiation defect of prechondrogenic cells in Bmpr1b-null phalanges. This finding indicates that BMP signaling is necessary for phalangeal prechondrogenic cells to differentiate into chondrocytes and that signaling of BMP receptor 1B in this context is replaceable by that of a constitutively active BMP receptor 1A. These results suggest that BMP signaling in prechondrogenic cells and in growth plate chondrocytes stimulates their chondrocytic differentiation and maturation toward hypertrophy, respectively.

Effect of Osteoblast-Targeted Expression of Bcl-2 in Bone: Differential Response in Male and Female Mice

Transgenic mice (Col2.3Bcl-2) with osteoblast-targeted human Bcl-2 expression were established. Phenotypically, these mice were smaller than their wildtype littermates and showed differential effects of the transgene on bone parameters and osteoblast activity dependent on sex. The net effect was an abrogation of sex differences normally observed in wildtype mice and an inhibition of bone loss with age. Ex vivo osteoblast cultures showed that the transgene had no effect on osteoblast proliferation, but decreased bone formation. Estrogen was shown to stimulate endogenous Bcl-2 message levels. These studies suggest a link between Bcl-2 and sex regulation of bone development and age-related bone loss. Whereas Bcl-2 has been shown to be an important regulator of apoptosis in development, differentiation, and disease, its role in bone homeostasis and development is not well understood. We have previously showed that the induction of glucocorticoid-induced apoptosis occurred through a dose-dependent decrease in Bcl-2. Estrogen prevented glucocorticoid-induced osteoblast apoptosis in vivo and in vitro by preventing the decrease in Bcl-2 in osteoblasts. Therefore, Bcl-2 may be an important regulator of bone growth through mechanisms that control osteoblast longevity and function. Col2.3Bcl-2 mice were developed carrying a 2.3-kb region of the type I collagen promoter driving 1.8 kb of human Bcl-2 (hBcl-2). Tissue specific expression of hBcl-2 in immunoassays validated the transgenic animal model. Histomorphometry and DXA were performed. Proliferation, mineralization, and glucocorticoid-induced apoptosis were examined in ex vivo cultures of osteoblasts. The effect of estrogen on mouse Bcl-2 in ex vivo osteoblast cultures was assayed by RT-PCR and Q-PCR. Two Col2.3Bcl-2 (tg/+) founder lines were established and appeared normal except that they were smaller than their nontransgenic wildtype (+/+) littermates at 1, 2, and 6 months of age, with the greatest differences at 2 months. Immunohistochemistry showed hBcl-2 in osteoblasts at the growth plate and cortical surfaces. Nontransgenic littermates were negative. Western blots revealed hBcl-2 only in type I collagen-expressing tissues. Histomorphometry of 2-month-old mice showed a significant decrease in tg/+ calvaria width with no significant differences in femoral trabecular area or cortical width compared with +/+. However, tg/+ males had significantly more trabecular bone than tg/+ females. Female +/+ mice showed increased bone turnover with elevated osteoblast and osteoclast parameters compared with +/+ males. Col2.3Bcl-2 mice did not show such significant differences between sexes. Male tg/+ mice had a 76.5 +/- 1.5% increase in ObS/BS with no significant differences in bone formation rate (BFR) or mineral apposition rate (MAR) compared with male +/+ mice. Transgenic females had a significant 48.4 +/- 0.1% and 20.1 +/- 5.8% decrease in BFR and MAR, respectively, compared with +/+ females. Osteoclast and osteocyte parameters were unchanged. By 6 months, femurs from female and male +/+ mice had lost a significant amount of their percent of trabecular bone compared with 2-month-old mice. There was little to no change in femoral bone in the tg/+ mice with age. Ex vivo cultures of osteoblasts from +/+ and Col2.3Bcl-2 mice showed a decrease in mineralization, no effect on proliferation, and an inhibition of glucocorticoid-induced apoptosis in Col2.3Bcl-2 cultures. Estrogen was shown to increase mouse Bcl-2 transcript levels in osteoblast cultures of wildtype mice, supporting a role for Bcl-2 in the sex-related differences in bone phenotype regulated by estrogen. Therefore, Bcl-2 differentially affected bone phenotype in male and female transgenic mice, altered bone cell activity associated with sex-related differences, and decreased bone formation, suggesting that apoptosis is necessary for mineralization. In addition, Bcl-2 targeted to mature osteoblasts seemed to delay bone development, producing a smaller transgenic mouse compared with wildtype littermates. These studies suggest that expression of Bcl-2 in osteoblasts is important in regulating bone mass in development and in the normal aging process of bone.

NFAT and Osterix cooperatively regulate bone formation

Immunosuppressants are crucial in the prevention of detrimental immune reactions associated with allogenic organ transplantation, but they often cause adverse effects in a number of biological systems, including the skeletal system. Calcineurin inhibitors FK506 and cyclosporin A inhibit nuclear factor of activated T cells (NFAT) activity and induce strong immunosuppression. Among NFAT proteins, NFATc1 is crucial for the differentiation of bone-resorbing osteoclasts. Here we show FK506 administration induces the reduction of bone mass despite a blockade of osteoclast differentiation. This reduction is caused by severe impairment of bone formation, suggesting that NFAT transcription factors also have an important role in the transcriptional program of osteoblasts. In fact, bone formation is inhibited in Nfatc1- and Nfatc2-deficient cells as well as in FK506-treated osteoblasts. Overexpression of NFATc1 stimulates Osterix-dependent activation of the Col1a1 (encoding type I collagen) promoter, but not Runx2-dependent activation of the Bglap1 (encoding osteocalcin) promoter. NFAT and Osterix form a complex that binds to DNA, and this interaction is important for the transcriptional activity of Osterix. Thus, NFAT and Osterix cooperatively control osteoblastic bone formation. These results may provide important insight into the management of post-transplantation osteoporosis as well as a new strategy for promoting bone regeneration in osteopenic disease.

Wnt‐mediated regulation of chondrocyte maturation: Modulation by TGF‐β

Wnt proteins are expressed during limb morphogenesis, yet their role and mechanism of action remains unclear during long bone growth. Wnt expression, effects and modulation of signaling events by BMP and transforming growth factor-beta (TGF-beta) were evaluated in chick embryonic chondrocytes. Chondrocyte cell cultures underwent spontaneous maturation with increased expression of colX and this was associated with an increase in the expression of multiple Wnts, including Wnt 4, 5a, 8c, and 9a. Both parathyroid hormone related peptide (PTHrP) and TGF-beta inhibited colX, but had disparate effects on Wnt expression. While TGF-beta strongly inhibited all Wnts, PTHrP did not inhibit either Wnt8c or Wnt9a and had lesser effects on the expression of the other Wnts. BMP-2 induced colX expression, and also markedly increased Wnt8c expression. Overexpression of beta-catenin and/or T cell factor (TCF)-4 also induced the type X collagen promoter. Overexpression of Wnt8c induced maturation, as did overexpression of beta-catenin. The Wnt8c/beta-catenin maturational effects were enhanced by BMP-2 and inhibited by TGF-beta. TGF-beta also inhibited activation of the Topflash reporter by beta-catenin, suggesting a direct inhibitory effect since the Topflash reporter contains only beta-catenin binding sequences. In turn beta-catenin inhibited activation of the p3TP-Luc reporter by TGF-beta, although the effect was partial. Thus, Wnt/beta-catenin signaling is a critical regulator of the rate of chondrocyte differentiation. Moreover, this pathway is modulated by members of the TGF-beta family and demonstrates the highly integrated nature of signals controlling endochondral ossification.

Expression of Cre recombinase in the mouse developing chondrocytes driven by the mouse ?2(XI) collagen promoter

Expression of Cre recombinase in the mouse developing chondrocytes driven by the mouse ?2(XI) collagen promoter

Akt Mediates Mechanical Strain-Induced Collagen Production by Mesangial Cells

Increased glomerular hydrostatic pressure is an important determinant of glomerulosclerosis and can be modeled by in vitro exposure of mesangial cells to cyclic mechanical strain. Stretched mesangial cells increase extracellular matrix protein production, the hallmark of glomerulosclerosis. Recent data indicate that the serine/threonine kinase Akt may be involved in matrix modulation. Thus, Akt activation and matrix synthesis in stretched mesangial cells were studied. Exposure of mesangial cells to 1 Hz cyclic strain led to prompt Akt activation, which was biphasic to 24 h. Activation was dependent on signaling through phosphatidylinositol-3-kinase and required EGF receptor transactivation. Inhibition of signaling through the PDGF receptor, Src kinase, or cytoskeletal disruption failed to prevent strain-induced Akt activation. Collagen type 1A1 transcript expression, promoter activation, and protein secretion were increased by stretch at 24 h and were dependent on phosphatidylinositol-3 kinase. Overexpression of dominant-negative Akt inhibited strain-induced collagen 1A1 production. Conversely, overexpression of constitutively active Akt led to increased collagen 1A1 upregulation and secretion. Finally, Akt activation was observed in the glomeruli of remnant rat kidneys, a model marked by increased intraglomerular pressure. The authors conclude that mechanical strain induces Akt activation in mesangial cells through a mechanism requiring phosphatidylinositol-3-kinase and EGF receptor transactivation. Type 1 collagen production is dependent on Akt and can be induced by Akt overexpression. Akt activation is observed in remnant kidneys in vivo. Thus, the role of Akt in progression of chronic hemodynamic glomerular disease is worthy of further exploration.

Fibroblast expression of the coactivator p300 governs the intensity of profibrotic response to transforming growth factor β

Transforming growth factor beta (TGFbeta) induces profibrotic responses in normal fibroblasts, and plays a fundamental role in the pathogenesis of fibrosis in scleroderma (systemic sclerosis [SSc]). The intensity of cellular responses elicited by cytokines is modulated by transcriptional coactivators such as the histone acetylase p300. The objective of these studies was to delineate the physiologic role of p300 in Smad-dependent profibrotic responses elicited by TGFbeta. Ectopic p300 was transiently expressed in normal dermal fibroblasts. Cellular p300 levels were suppressed using p300-specific ribozymes. The regulation of gene expression was examined by transient transfection assays, Northern blotting, and immunoblot analysis. The expression of p300 in normal and scleroderma fibroblasts was evaluated by confocal microscopy and immunoblotting, and p300 levels in skin from mice with experimental scleroderma were assessed by immunohistochemistry. In normal fibroblasts, TGFbeta induced an increase in the levels of p300. Forced expression of ectopic p300 in these cells dramatically enhanced the magnitude of TGFbeta responses, whereas selective depletion of p300 using ribozyme resulted in abrogation of TGFbeta-induced collagen synthesis and promoter activity. Furthermore, TGFbeta lost its ability to induce Smad-dependent transcription in p300-depleted fibroblasts. These responses could be fully rescued with ectopic p300. Abrogation of Smad-mediated TGFbeta signaling was not due to alterations in the levels or the ligand-dependent phosphorylation or intracellular trafficking of endogenous Smads. Immunohistochemical analysis demonstrated substantially increased p300 expression in lesional skin from mice with chronic graft-versus-host disease, an animal model of scleroderma. Furthermore, levels of p300 were 2-3-fold higher in cultured fibroblasts derived from SSc patients than in fibroblasts from matched normal controls. These results establish, for the first time, that the coactivator histone acetylase p300, itself a target of TGFbeta regulation, is an essential component of the cellular TGFbeta signal transduction pathways mediating stimulation of collagen synthesis in fibroblasts. Since the cellular abundance of p300 appears to govern the intensity of profibrotic responses elicited by TGFbeta, elevated p300 expression in lesional tissue may contribute to the progression of skin fibrosis in scleroderma.

B-Myb Represses Elastin Gene Expression in Aortic Smooth Muscle Cells

B-Myb represses collagen gene transcription in vascular smooth muscle cells (SMCs) in vitro and in vivo. Here we sought to determine whether elastin is similarly repressed by B-Myb. Levels of tropoelastin mRNA and protein were lower in aortas and isolated SMCs of adult transgenic mice expressing the human B-myb gene, driven by the basal cytomegalovirus promoter, compared with age-matched wild type (WT) animals. However, the vessel wall architecture and levels of insoluble elastin revealed no differences. Since elastin deposition occurs early in development, microarray analysis was performed using nontransgenic mice. Aortic levels of tropoelastin mRNA were low during embryonal growth and increased substantially in neonates, whereas B-myb levels varied inversely. Tropoelastin mRNA expression in aortas of 6-day-old neonatal transgenic and WT animals was comparable. Recently, we demonstrated that cyclin A-Cdk2 prevents B-Myb-mediated repression of collagen promoter activity. Cyclin A2 levels were higher in neonatal versus adult WT or transgenic mouse aortas. Ectopic cyclin A expression reversed the ability of B-Myb to repress elastin gene promoter activity in adult SMCs. These results demonstrate for the first time that B-Myb represses SMC elastin gene expression and that cyclin A plays a role in the developmental regulation of elastin gene expression in the aorta. Furthermore, the findings provide additional insight into the mechanism of B-myb-mediated resistance to femoral artery injury.

2(I) collagen gene regulation by protein kinase C signaling in human dermal fibroblasts

We investigated the mechanisms by which protein kinase C (PKC) regulates the expression of the α2(I) collagen gene in normal dermal fibroblasts. Reduction of PKC-α activity by treatment with Gö697-6 or by overexpression of a dominant negative (DN) mutant form decreased α2(I) collagen gene expression. This decrease required a sequence element in the collagen promoter that contains Sp1/Sp3 binding sites. Reduction of PKC-δ activity by rottlerin or overexpression of DN PKC-δ also decreased α2(I) collagen gene expression. This effect required a separate sequence element containing Sp1/Sp3-binding sites and an Ets-binding site. In both cases, point mutations within the response elements abrogated the response to PKC inhibition. Forced overexpression of Sp1 rescued the PKC inhibitor-mediated reduction in collagen protein expression. A DNA affinity precipitation assay revealed that inhibition of PKC-δ by rottlerin increased the binding activity of endogenous Fli1 and decreased that of Ets1. On the other hand, TGF-β1, which increased the expression of PKC-δ, had the opposite effect, increasing the binding activity of Ets1 and decreasing that of Fli1. Our results suggest that PKC-δ is involved in the regulation of the α2(I) collagen gene in the presence or absence of TGF-β. Alteration of the balance of Ets1 and Fli1 may be a novel mechanism regulating α2(I) collagen expression.

Pathogenesis of Disturbed Calcium Phosphate Metabolism in Renal Failure-We Have to Rewrite the Textbooks: Transgenic Mice Overexpressing Human Fibroblast Growth Factor 23 (R176Q) Delineate a Putative Role for Parathyroid Hormone in Renal Phosphate Wasting Disorders. Endocrinology 145: 5269-5279, 2004.

In the past, nephrologists were aware of the heritable disorders of renal phosphate wasting, i.e., X-linked hypophosphatemia (XLH) and the rare autosomal dominant hypophosphatemic rickets (ADHR), as well as tumor-induced osteomalacia (1), long considered exotic and rare conditions of limited practical interest. All this has dramatically changed with the unraveling of the underlying genetics. A mutation of a protease (PHEX) was identified in XLH and in ADHR, a mutation of fibroblast growth factor 23 (FGF23) located in a sequence motif crucial for cleavage by furin proteases. The exact role of PHEX is still controversial (2) and additional molecules of unknown pathogenetic significance have been identified, i.e., the phosphaturic frizzled related protein (FRP4) and matrix extracellular phosphoglycoprotein of unknown function (3). The crucial breakthrough that got this field moving has been the cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia by Shimada et al. (4). Native as well as mutated FGF23 suppresses Na+-dependent phosphate cotransport in proximal tubules (5) and—somewhat unanticipated—also reduces the 1-α hydroxylase activity as well as circulating 1,25(OH)2D3 concentrations. Recombinant FGF23 carrying the ADHR mutation causes phosphaturia and hypophosphatemia (6), and conversely targeted ablation of the FGF23 gene causes hyperphosphatemia (7), documenting the key importance of this molecule in the maintenance of normal phosphate concentrations. For nephrologists the scene has become exciting with the observation in animals and patients with impaired renal function that the concentrations of the phosphaturic “hormone” FGF23 are elevated (8–10), although some methodological points remain to be clarified. The unresolved issue remained: Was oversecretion of FGF23 just an appropriate response to phosphate retention, or was FGF23 a primary mover in renal failure provoking the known endocrine abnormalities of hyperparathyroidism and deficiency of active vitamin D? The latter possibility is rendered increasingly more plausible by recent studies. Transgenic mice expressing FGF23 under the control of an α1(I)collagen promoter in bone had low serum-P concentrations, high urinary P excretion, and high parathyroid hormone (PTH) concentrations, while there were no differences in serum creatinine and 1,25(OH)2D3 (11). Even more suggestive are the results in a model where FGF23 was overexpressed in the liver under the influence of apolipoprotein E3 as a promoter. FGF23 carried a mutation (R176Q) which protected against enzymatic degradation. Such massive overproduction of less biodegradable FGF23 caused not only hypophosphatemia and phosphaturia, but also low 1,25(OH)2D3 and massively elevated PTH concentrations associated with severe bone lesions (rickets and osteomalacia). The observation that high FGF23 causes hyperparathyroidism in the absence of renal malfunction, either directly or indirectly via lower 1,25(OH)2D3 concentrations, is obviously tantalizing in view of the question whether high FGF23 concentrations in renal failure are just an innocent reaction to hyperphosphatemia or driving the endocrine abnormalities of PTH oversecretion and 1,25(OH)2D3 underproduction. In this context it is of great interest that long ago high PTH had been noted in patients with ADHR despite normal renal function and before any therapeutic administration of phosphate (12,13). The same was seen in XLH (14). This is a rapidly moving frontier and the nephrologist is well advised to stay tuned. It is too early in the day to draw a definite pathogenetic scheme, but it is likely that in the genesis of hyperparathyroidism of renal failure, the classical trio of low Ca, high P, and low active D will now become a quartet including FGF23 as an active player.

Ischemia can stimulate the collagen promoter activity in the huma hepatic stellate cell line, LI-90: Kozo Kayano, Makoto Segawa, Eiki Sakaguchi, Isao Sakaida, Kiwamu Okita, Yamaguchi University School of Medicine, Ube City Yamaguchi Prefecture Japan

Ischemia can stimulate the collagen promoter activity in the huma hepatic stellate cell line, LI-90: Kozo Kayano, Makoto Segawa, Eiki Sakaguchi, Isao Sakaida, Kiwamu Okita, Yamaguchi University School of Medicine, Ube City Yamaguchi Prefecture Japan

Differential effects of ERK and p38 signaling in BMP-2 stimulated hypertrophy of cultured chick sternal chondrocytes

BackgroundDuring endochondral bone formation, the hypertrophy of chondrocytes is accompanied by selective expression of several genes including type X collagen and alkaline phosphatase. This expression is stimulated by inducers including BMPs and ascorbate. A 316 base pair region of the type X collagen (Col X) promoter has been previously characterized as the site required for BMP regulation. The intent of this study was to examine the role of Mitogen Activated Protein (MAP) and related kinase pathways in the regulation of Col X transcription and alkaline phosphatase activity in pre-hypertrophic chick chondrocytes.ResultsUsing a luciferase reporter regulated by the BMP-responsive region of the type X collagen promoter, we show that promoter activity is increased by inhibition of extra-cellular signal regulated kinases 1 or 2 (ERK1/2). In contrast the ability of BMP-2 to induce alkaline phosphatase activity is little affected by ERK1/2 inhibition. The previously demonstrated stimulatory affect of p38 on Col X was shown to act specifically at the BMP responsive region of the promoter. The inhibitory effect of the ERK1/2 pathway and stimulatory effect of the p38 pathway on the Col X promoter were confirmed by the use of mutant kinases. Inhibition of upstream kinases: protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase pathways increased basal Col X activity but had no effect on the BMP-2 induced increase. In contrast, ascorbate had no effect on the BMP-2 responsive region of the Col X promoter nor did it alter the increase in promoter activity induced by ERK1/2 inhibition. The previously shown increase in alkaline phosphatase activity induced by ascorbate was not affected by any kinase inhibitors examined. However some reduction in the alkaline phosphatase activity induced by the combination of BMP-2 and ascorbate was observed with ERK1/2 inhibition.ConclusionOur results demonstrate that ERK1/2 plays a negative role while p38 plays a positive role in the BMP-2 activated transcription of type X collagen. This regulation occurs specifically at the BMP-2 responsive promoter region of Col X. Ascorbate does not modulate Col X at this region indicating that BMP-2 and ascorbate exert their action on chondrocyte hypertrophy via different transcriptional pathways. MAP kinases seem to have only a modest effect on alkaline phosphatase when activity is induced by the combination of both BMP-2 and ascorbate.

Peroxisome Proliferator-activated Receptor γ Suppresses Proximal α1(I) Collagen Promoter via Inhibition of p300-facilitated NF-I Binding to DNA in Hepatic Stellate Cells

Peroxisome Proliferator-activated Receptor γ Suppresses Proximal α1(I) Collagen Promoter via Inhibition of p300-facilitated NF-I Binding to DNA in Hepatic Stellate Cells

Leptin enhances α1(I) collagen gene expression in LX-2 human hepatic stellate cells through JAK-mediated H2O2-dependent MAPK pathways

Leptin, a liver profibrogenic cytokine, induces oxidative stress in hepatic stellate cells (HSCs), with increased formation of the oxidant H2O2, which signals through p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, stimulating tissue inhibitor of metalloproteinase-1 production. Since oxidative stress is a pathogenic mechanism of liver fibrosis and activation of collagen gene is a marker of fibrogenesis, we evaluated the effects of leptin on collagen I expression. We report here that, in LX-2 human HSCs, leptin enhances the levels of alpha1(I) collagen mRNA, promoter activity and protein. Janus kinase (JAK)1 and JAK2 were activated. H2O2 formation was increased; this was prevented by the JAK inhibitor AG490, suggesting a JAK-mediated process. ERK1/2 and p38 were activated, and the activation was blocked by catalase, consistent with an H2O2-dependent mechanism. AG490 and catalase also prevented leptin-stimulated alpha1(I) collagen mRNA expression. PD098059, an ERK1/2 inhibitor, abrogated ERK1/2 activation and suppressed alpha1(I) collagen promoter activity, resulting in mRNA down-regulation. The p38 inhibitor SB203580 and overexpression of dominant negative p38 mutants abrogated p38 activation and down-regulated the mRNA. While SB203580 had no effect on the promoter activity, it reduced the mRNA half-life from 24 to 4 h, contributing to the decreased mRNA level. We conclude that leptin stimulates collagen production through the H2O2-dependent and ERK1/2 and p38 pathways via activated JAK1 and JAK2. ERK1/2 stimulates alpha1(I) collagen promoter activity, whereas p38 stabilizes its mRNA. Accordingly, interference with leptin-induced oxidative stress by antioxidants provides an opportunity for the prevention of liver fibrosis.

Transgenic mice that express Cre recombinasein hypertrophic chondrocytes

In order to investigate the physiological control of hypertrophic chondrocytes which present the terminally differentiated form of chondrocytes, we generated a mouse line expressing the Cre recombinase under the control of the mouse type X collagen (Col10a1) promoter. In situ hybridization analysis demonstrated the expression of Col10a1-Cre transgene in hypertrophic chondrocytes of femur at postnatal day 2 (P2). In order to test the excision activity of the Cre recombinase, the Col10a1-Cre transgenic line was crossed with the mouse strain carrying the Smad4 conditional alleles (Smad4co/co) and the reporter line ROSA26. Multiple tissue PCR of Col10a1-Cre;Smad4co/+ mice revealed the restricted Cre activity in tissues containing hypertrophic chondrocytes. LacZ staining revealed that the Cre activity was observed in the cartilage primordia of ribs at E14.5 and only detected in the lower hypertrophic region of ribs at P1. These data suggest that the Col10a1-Cre mouse line described here could be used to achieve conditional gene targeting in hypertrophic chondrocytes.

A Role for the Androgen Receptor in Collagen Content of the Skin

Collagen, the major macromolecular component of skin, is responsible for maintaining the structural integrity of the tissue as well as for providing important functional characteristics, such as pliability and thickness. We have been studying the structure and regulation of collagen in mouse mutations affecting the skin. In the course of these studies, we found that there are significant differences in collagen content between the skin of wild-type male and female mice, which become evident at puberty. Furthermore, male mice with an X-linked mutation in the androgen receptor gene (formerly called testicular feminization and abbreviated as Ar(Tfm)) showed decreased levels of collagen, indicating that the androgen receptor pathway contributes to the observed differences. These findings demonstrate that there are striking differences in the collagen content of skin between male and female mice, and provide a biochemical explanation for these differences.

EFFECTS OF ACETALDEHYDE AND TNFα ON THE INHIBITORY KAPPA B- α PROTEIN AND NUCLEAR FACTOR KAPPA B ACTIVATION IN HEPATIC STELLATE CELLS

Increased plasma tumour necrosis alpha (TNFalpha) and elevated monocyte nuclear factor kappa B (NF-kappaB) are associated with liver injury and inflammation in models of alcoholic liver disease and are found to be elevated in monocytes of patients with alcoholic hepatitis. Acetaldehyde enhances, whereas TNFalpha inhibits, transcription of the type I collagen promoters and type I collagen production. NF-kappaB, an inhibitor of the type I collagen promoters, is increased by both acetaldehyde and TNFalpha. This study determined the effects of acetaldehyde in comparison to the effects of TNFalpha on inhibitory kappa B-alpha (IkappaB-alpha) protein and NF-kappaB activation in hepatic stellate cells. Activated rat hepatic stellate cells in culture were exposed to acetaldehyde or TNFalpha for short periods of time, following which the cells were harvested for the determination of IkappaB-alpha protein, IkappaB-alpha kinase activity and nuclear NF-kappaB. Acetaldehyde increased IkappaB-alpha kinase activity and decreased IkappaB-alpha after 10 min of exposure, with recovery towards control levels at 20 min. In contrast, TNFalpha resulted in higher IkappaB-alpha kinase activity at 20 min than at 10 min, and similar low IkappaB-alpha at 10 and 20 min. Both acetaldehyde and TNFalpha enhanced nuclear NF-kappaB (p65), but acetaldehyde alone also increased NF-kappaB (p50). TNFalpha and acetaldehyde independently activate NF-kappaB by rapid enhancement of IkappaB-alpha kinase activity and degradation of IkB-alpha protein. Increased TNFalpha is the principal mechanism for the elevation of NF-kappaB in severe alcoholic hepatitis. The elevation of NF-kappaB due to TNFalpha enhance liver injury, but inhibit fibrogenesis. In contrast, the effect of acetaldehyde in activating NF-kappaB is associated with increases in both liver injury and fibrogenesis, indicating that the effects of acetaldehyde on fibrogenesis are mediated by cytokines and by trans-acting factors other than NF-kappaB.

Major Histocompatibility Class II Transactivator (CIITA) Mediates Repression of Collagen (COL1A2) Transcription by Interferon γ (IFN-γ)

Interferon gamma (IFN-gamma) plays an important role during inflammation by repressing collagen and activating major histocompatibility class II (MHC-II) expression. Activation of MHC-II by IFN-gamma requires regulatory factor for X-box 5 (RFX5) complex as well as class II transactivator (CIITA). We have shown that the RFX family binds to the COL1A2 transcription start site, and the RFX5 complex represses COL1A2 gene expression during IFN-gamma response. In this report, we demonstrate that CIITA is a key mediator of COL1A2 repression by IFN-gamma. IFN-gamma up-regulates the expression of CIITA in a time-dependent manner in lung fibroblasts and promotes CIITA protein occupancy on COL1A2 transcription start site in vivo as judged by chromatin immunoprecipitation (ChIP) assays. There are coordinate decreases in the occupancy of RNA polymerase II on the collagen transcription start site with increasing CIITA occupancy during IFN-gamma treatment. In addition, we are able to specifically knockdown the IFN-gamma-stimulated expression of CIITA utilizing short hairpin interference RNA (shRNA) against CIITA. This leads to the alleviation of COL1A2 repression and MHC-II activation by IFN-gamma. RFX5 recruits CIITA to the collagen site as evidenced by DNA affinity chromatography. The presence of RFX5 complex proteins enhances the collagen repression by CIITA reaching levels occurring during IFN-gamma treatment. Co-expression of CIITA with deletion mutations and collagen promoter constructs demonstrates that CIITA represses collagen promoter mainly through its N-terminal region including the acidic domain and the proline/serine/threonine domain. Our data suggest that CIITA is a crucial member of a repressor complex responsible for mediating COL1A2 transcription repression by IFN-gamma.

High ambient glucose enhances sensitivity to TGF-beta1 via extracellular signal--regulated kinase and protein kinase Cdelta activities in human mesangial cells.

High ambient glucose activates intracellular signaling pathways to induce cytokines such as TGF-beta1 in the extracellular matrix accumulation of diabetic nephropathy. These same pathways also may directly modulate TGF-beta1 signaling. R-Smad phosphorylation, association with Smad4, and nuclear accumulation after TGF-beta1 treatment (1.0 ng/ml) were significantly higher in mesangial cells that were conditioned to 20 mM glucose for 72 h than mesangial cells in 6.5 mM glucose, suggesting that high glucose enhanced responsiveness to TGF-beta1. Neither TGF-beta1 bioactivity nor TGF-beta receptor binding was significantly different between in 6.5 and 20 mM glucose-conditioned cultures. Furthermore, adding a neutralizing anti-TGF-beta1 antibody during glucose conditioning did not affect the enhanced Smad responsiveness, indicating that enhancement likely did not result from increased TGF-beta expression. In contrast, a mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK inhibitor, PD98059, completely abrogated the effect of high glucose. Glucose stimulation of ERK was inhibited by the general protein kinase C (PKC) inhibitor calphostin C and by the PKCdelta-specific inhibitor rottlerin, whereas Gö6976, an inhibitor of conventional PKC, had no effect on ERK activity. Specificity of the PKC inhibitors was further verified by PKCbeta and delta kinase assay. High glucose increased expression of several PKC isozymes, but only PKCdelta showed proportionally increased membrane translocation and kinase activity in cells that were conditioned to 20 mM glucose. Finally, both ERK and PKCdelta inhibition during glucose conditioning abrogated enhanced alpha1(I) collagen mRNA and promoter induction by TGF-beta1. Taken together, these data strongly suggest that heightened ERK and PKCdelta activity in high ambient glucose conditions interact with the Smad pathway, leading to enhanced responsiveness to TGF-beta1 and increased extracellular matrix production in mesangial cells.

Expression of genes for collagen production and NF-κB gene activation of in vivo healing flexor tendons

Purpose Collagen production and reorientation are core events in the healing of tendons and are essential for gaining tensile strength. Intrasynovial flexor tendons are composed chiefly of type I collagen; however, type III collagen is the earliest to be laid down in the wounds. We investigated the expression of genes for type I collagen, type III collagen and its promoter, and nuclear transcription factor kappa B (NF-κB) in an in vivo tendon injury model in chickens. Methods Flexor digitorum profundus tendons of the long toes of 12 white leghorn chickens were transected partially and repaired with the modified Kessler method. The repaired tendon segments were harvested at 2, 4, and 8 weeks after surgery with 4 chickens used each time. The uninjured tendons from 4 other chickens were used as the controls. We determined the expression of the collagen and NF-κB genes in the tendons with reverse transcription and polymerase chain reaction and semiquantitative analysis of gene bands in gel electrophoresis. Results Expression of the type III collagen gene was enhanced significantly in the tendons at 2, 4, and 8 weeks after repair. Levels of expression of the type III collagen promoter gene were increased significantly in the healing tendons at 2 and 4 weeks. Increases in the expression of the type I collagen gene occurred later than those of the type III collagen and NF-κB genes and were significant in the tendons after 4 and 8 weeks. The NF-κB gene was activated in the tendons at all the healing periods. Conclusions Expression of type III collagen and its promoter genes is enhanced remarkably during in vivo flexor tendon healing. The NF-κB gene is activated during the healing process. Expression of the type I collagen gene increases remarkably at later periods of in vivo tendon healing and is proportional to that of the NF-κB gene.