Biglycan mRNA Research Articles

Intervertebral disc degeneration and ectopic bone formation in apolipoprotein E knockout mice

Cardiovascular risk factors are known to be associated with intervertebral disc degeneration, but the underlying mechanism is still unclear. The ApoE knockout (KO) mouse is a well-established model for atheroscelorosis. We hypothesized that ApoE is involved in maintaining disc health and that ApoE KO mice will develop early disc degeneration. Discs of ApoE KO and wild-type (WT) mice were characterized with histological/immunological, biochemical, and real-time RT-PCR assays. A comparison of the extracellular matrix production was also performed in disc cells. We demonstrated that ApoE was highly expressed in the endplates of WT discs, and ectopic bone formed in the endplates of ApoE KO discs. Glycosaminoglycan content was decreased in both ApoE KO annulus fibrosus (AF) and nucleus pulposus (NP) cells. Collagen levels were increased in AF and decreased in NP cells. Matrix metalloproteinase-3, -9, and -13 expressions were increased, which may partially explain the impaired matrix production. We also found collagen I, II, aggrecan, and biglycan mRNA expressions were increased in AF cells but decreased in NP cells. Apoptosis was increased in the ApoE KO NP tissue. These results suggest early disc degeneration changes in the ApoE KO mice. ApoE may play a critical role in disc integrity and function.

The small leucine-rich proteoglycan, biglycan, is highly expressed in adipose tissue of Psammomys obesus and is associated with obesity and type 2 diabetes

We have previously demonstrated that the small leucine-rich proteoglycan decorin may play a role in adipose tissue homeostasis and the pathophysiology of obesity. Biglycan is highly similar in structure to decorin, therefore we hypothesized it would have a similar expression profile and role to decorin in adipose tissue. Real time polymerase chain reaction was used to measure biglycan mRNA levels in adipose tissue from normal glucose tolerant and impaired glucose tolerant and type 2 diabetic (T2D) Psammomys obesus. Biglycan mRNA was found to be highly expressed in adipose tissue, and gene expression was significantly higher in visceral compared to subcutaneous adipose tissue, with elevated levels in obese, T2D compared to lean normal glucose tolerant P. obesus (P < 0.04). Biglycan mRNA was predominantly expressed by stromal/vascular cells of fractionated adipose tissue (P = 0.023). Biglycan expression in adipose tissue, particularly in the obese state, was markedly upregulated. Collectively, our data suggest that the small leucine-rich proteoglycan family proteins biglycan and decorin may play a role in the development of obesity and T2D, possibly by facilitating expansion of adipose tissue mass.

Myostatin regulates proliferation and extracellular matrix mRNA expression in NIH3T3 fibroblasts

The aim of this study was to clarify the effects of myostatin, which is a negative regulator of skeletal muscle mass, on the proliferation of NIH3T3 fibroblasts and the synthesis of extracellular matrix (ECM) by them. A proliferation assay revealed that myostatin attenuated cell growth at any of the doses used. High doses of myostatin strongly inhibited cell proliferation. Moreover, myostatin receptor, activin receptor type-2B (ActRIIB), was found to be distributed on cells and it was also clarified that myostatin increased the expression of cyclin-dependent kinase inhibitor p21 (p21). These results suggested that a high dose of myostatin inhibits fibroblast proliferation by the same mechanism as that for inhibition of myoblast proliferation. We then examined the effects of myostatin on the mRNA expression of ECM molecules (decorin, biglycan, type I collagen, type III collagen, type IV collagen and type V collagen) by real-time PCR. Real-time PCR showed that myostatin increased the mRNA of decorin, biglycan and collagen (types I, IV and V) in fibroblasts. The results suggest that myostatin regulates ECM synthesis in cultured fibroblasts.

Biglycan expression correlates with aggressiveness and poor prognosis of gastric cancer

Biglycan, a member of the small leucine-rich proteoglycan family, has been implicated in the development and progression of human cancers. However, the clinical significance of biglycan expression in gastric cancer has not been determined. In the present study, biglycan mRNA and protein concentrations were analyzed using quantitative realtime reverse transcription polymerase chain reaction and Western blot in 69 gastric cancer and adjacent non-tumorous tissues, respectively. Biglycan expression was further assessed using immunohistochemistry in tissue microarrays that contained 264 cases of gastric cancer, and others containing normal or metastasized lymph node tumor tissues. Biglycan was upregulated at the transcriptional and translational levels and there was a correlation between the expression of biglycan mRNA and protein (P = 0.000, κ = 0.769). Over-expression of biglycan was strongly associated with lymph node metastasis, tumor (T) classification, metastasis (M) classification, vascular invasion and Union for International Cancer Control (UICC) stage. Patients with biglycan-positive tumors had a significantly higher disease recurrence rate and poorer survival than patients with biglycan-negative tumors after the radical surgery. Multivariate analysis revealed that biglycan expression is an independent prognostic indicator for survival of patients with gastric cancer. The data from the current study demonstrate that elevated expression of biglycan may play an important role in the development and progression of gastric cancer, and could be further evaluated as a biomarker for predication of a poor clinical outcome.

TGF-β stimulates biglycan core protein synthesis but not glycosaminoglycan chain elongation via Akt phosphorylation in vascular smooth muscle

Transforming growth factor-β (TGF-β) can mediate proteoglycan synthesis via Smad and non-Smad signalling pathways in vascular smooth muscle (VSM). We investigated whether TGF-β-mediated proteoglycan synthesis is via PI3K/Akt. TGF-β induced a rapid phosphorylation of Akt that continued upto 4 h. Akt phosphorylation was blocked by Akt1/2 inhibitor SN30978; however, it did not block Smad2 phosphorylation at either the carboxy or linker regions indicating that TGF-β-mediated Akt phosphorylation is independent of Smad2 signalling. The role of Akt in TGF-β-mediated proteoglycan synthesis was investigated. Treatment with SN30978 showed a concentration-dependent decrease in TGF-β-mediated [35S]-sulphate and [35S]-Met/Cys incorporation into secreted proteoglycans; however, SDS-PAGE showed no change in biglycan size. In TGF-β-treated cells, biglycan mRNA levels increased by 40–100% in 24 h and was significantly blocked by SN30978. Our findings demonstrate that Akt is a downstream signalling component of TGF-β-mediated biglycan core protein synthesis but not glycosaminoglycan chain hyper-elongation in VSM.

Up-regulated biglycan expression correlates with the malignancy in human colorectal cancers

Biglycan, an extracellular matrix protein, has been implicated in the oncogenesis and cancer development in various types of human cancer. The clinical significance of biglycan in colorectal cancer, however, remains unclear. In the present study, biglycan mRNA expression was analyzed in 110 samples (primary colorectal tumor and matched adjacent normal tissue) derived from 55 patients with colorectal cancer using quantitative real-time RT-PCR. The correlations between biglycan up-regulation and the clinicopathological data were also evaluated. We found that the up-regulation of biglycan occurred in 61.8% (34/55) of colorectal cancer tissues, and biglycan expression in colorectal cancer tissues was markedly higher than that in corresponding normal tissues (P = 0.0264). Moreover, statistical analysis displayed a significant correlation in biglycan up-regulation with poor tumor differentiation (P = 0.009), lymph node metastasis (P = 0.041), and distant metastasis (P = 0.036). However, there was no significant correlation between biglycan up-regulation and other clinicopathological factors (all P > 0.05). In conclusion, biglycan may be a potential marker for the malignancy of colorectal cancer.

Acute inflammation plays a limited role in the regulation of adipose tissue COL1A1 protein abundance

Obesity is an inflammatory condition that is also associated with increased extracellular matrix (ECM) gene expression. However, a direct link between adipose tissue inflammation and ECM gene expression has not been established. Therefore, we determined the effect of chronic inflammation induced by obesity and acute inflammation by lipopolysaccharide (LPS) challenge on ECM genes including biglycan (BGN), collagen 1A1 (COL1A1) and COL6A1, major ECM genes in adipose tissue. Male C57BL/6J mice fed either a control diet (10% fat calories) or a high-fat diet (HFD) (60% fat calories) for 6 weeks were treated with LPS or saline 24 h before sacrifice. Expression of ECM genes in the epididymal (EWAT) and subcutaneous adipose tissue (SWAT) was determined by RT-PCR and protein abundance by Western blotting. Human SWAT from lean and obese subjects was also analyzed. Increased messenger RNA (mRNA) expression of ECM genes BGN and COL1A1 was observed in the mouse EWAT after HFD (P<.05). However, reduced amount of COL1A1 protein was observed in EWAT of mice on HFD and in SWAT from obese human subjects. Acute inflammation induced BGN mRNA in EWAT, enhanced the gene expression of matrix metalloproteases (MMPs) 3 and 9. Acute inflammation also resulted in higher MMP9 gelatinolytic activity; however, this showed no association with COL1A1 protein abundance. Higher MMP2 expression in mice on HFD suggests its involvement in the reduction of COL1A1 protein abundance with HFD. Elevated MMP9 gelatinolytic activity in SWAT from obese humans indicates a prominent role for MMP9 in SWAT COL1A1 protein turnover in humans.

Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone

BackgroundWe have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues.MethodsE2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues.ResultsDecorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition.ConclusionsThe patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.

Effects of the angiotensin II receptor blocker losartan on the monocyte expression of biglycan in hypertensive patients

1. Recently, we demonstrated that biglycan (BGN) is increased in circulating monocyte cells from hypertensive patients and that angiotensin (Ang) II is able to increase BGN expression. The present study was designed to investigate the effects of treatment with the angiotensin AT(1) receptor antagonist losartan on monocyte BGN mRNA and protein expression in essential hypertension. 2. One hundred and twenty-six newly diagnosed hypertensive patients without additional risk factors for atherosclerosis and cardiovascular disease were treated with 100 mg losartan once daily for 6 months. Biglycan mRNA and protein expression was determined in monocytes isolated from peripheral blood before (T(0)) and after (T(1)) therapy. Plasma levels of interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and high sensitivity C-reactive protein (hs-CRP) were also determined. In addition, BGN mRNA and protein expression was determined after the ex vivo addition of 1 micromol/L AngII to monocytes isolated from 20 randomly selected hypertensive patients. 3. Biglycan mRNA and protein expression, blood pressure and plasma levels of fibrinogen, IL-6, TNF-alpha and CRP were significantly lower at T(1) than at T(0). Variations in BGN expression were associated with inflammatory markers, but not directly with blood pressure. In AngII-stimulated monocytes, BGN mRNA and protein expression was significantly lower at T(1) that at T(0). Moreover, mean BGN mRNA expression in AngII-stimulated monocytes isolated from losartan-treated patients was similar to baseline expression in unstimulated monocytes from untreated patients. 4. The results of the present study show that losartan can reduce BGN expression in monocytes from hypertensive patients, without any linear association with blood pressure, suggesting that the effects of AngII on BGN expression in monocytes may be modulated, in part, by an AT(1) receptor blocker.

In Vivo Low-Intensity Pulsed Ultrasound (LIPUS) Following Tendon Injury Promotes Repair During Granulation but Suppresses Decorin and Biglycan Expression During Remodeling

Bench research, cross-sectional. To determine if the effects of low-intensity pulsed ultrasound (LIPUS) on matrix synthesis change at different stages of tendon healing. LIPUS is effective in promoting tendon healing by stimulation of matrix synthesis. The timing of initiation and duration of LIPUS treatment have been shown to affect its effectiveness to promote tendon healing, suggesting a change of tissue responses to LIPUS stimulation. Understanding how the cellular responses to LIPUS stimulation change during tendon healing is thus important. In a rat model of patellar tendon donor site injury, a single sonication of LIPUS or mock sonication was delivered to the injured knee of the rats on the fourth, 14th or 28th day postinjury. Tendon samples were harvested at 4 hours and 24 hours after sonication and the mRNA expression of COL1A1, COL3A1, decorin, biglycan, and TGF-beta 1 was analyzed. The results showed that a single sonication of LIPUS increased COL1A1 and COL3A1 mRNA in healing patellar tendons when administered on the fourth or 14th day postinjury, but not when administered on the 28th day postinjury. Both decorin and biglycan mRNA were decreased by treatment with LIPUS on the 28th day postinjury. Our results showed that LIPUS enhanced collagen synthesis in vivo only during the granulation phase. Matrix remodeling may be affected by LIPUS with the suppressed expression of decorin and biglycan. Our findings suggest that LIPUS should be applied during the granulation phase but not during the remodeling phase, to promote tendon healing.

Placental Biglycan Expression is Decreased in Human Idiopathic Fetal Growth Restriction

Fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. The majority of FGR cases are idiopathic and are associated with placental insufficiency, which can result from placental thrombosis. Evidence suggests that Dermatan Sulfate (DS) is an important anti-coagulant in placentae of uncomplicated pregnancies. This study hypothesised that the expression of biglycan proteoglycan, a source of DS, is decreased in idiopathic FGR placentae compared with placentae from uncomplicated pregnancies. This study aimed to determine biglycan mRNA, protein expression and spatial distribution in idiopathic FGR placentae compared with the placentae from gestation-matched controls. Biglycan mRNA expression, protein expression and spatial distribution was determined in 26 idiopathic FGR-affected placentae and 27 placentae from gestation-matched controls (27–40 weeks gestation) using real-time PCR, immunoblotting and immunohistochemistry, respectively. Mean biglycan mRNA expression was significantly decreased in FGR placentae compared with control placentae (2.87 ± 0.55, ( n = 26) vs. 4.48 ± 0.85, ( n = 27); t-test p = 0.01). FGR placentae demonstrated a trend towards decrease in mean biglycan protein expression compared with control placentae (0.86 ± 0.22 ( n = 9, FGR) vs, 1.9 ± 0.56 ( n = 7, control) p = 0.07). Biglycan immunoreactivity was detected in endothelial cells and sub-endothelial cells of the perivascular region of fetal capillaries. Semi-quantitative analyses demonstrated a significant decrease in immunoreactive biglycan in FGR placentae compared with control placentae (51.1 ± 19.3 vs, 500.7 ± 223, n = 6, p < 0.001). This is the first study to demonstrate decreased biglycan expression in idiopathic FGR placentae compared to gestation-matched controls. Reduced biglycan expression may contribute to placental thrombosis within the fetal vasculature, and may contribute to the pathogenesis of idiopathic FGR.

Transcriptional and posttranscriptional regulators of biglycan in cardiac fibroblasts

Biglycan, a small leucine-rich proteoglycan, is essential for scar formation and preservation of hemodynamic function after myocardial infarction, as shown in biglycan-knockout mice. Because of this important role in cardiac pathophysiology, we aimed to identify regulators of biglycan expression and posttranslational modifications in cardiac fibroblasts. Cardiac fibroblasts were isolated from neonatal Wistar-Kyoto rats and used in the first passage. Expression of biglycan was analyzed after metabolic labeling with [(35)S]-sulfate by SDS-polyacrylamide gel electrophoresis and molecular sieve chromatography; mRNA expression was examined by Northern analysis and real-time RT-PCR. Serum, thrombin, transforming growth factor beta1 (TGFbeta 1) and platelet-derived growth factor BB (PDGF-BB) strongly increased [(35)S]-labeled proteoglycan levels. Tumor necrosis factor alpha further increased the stimulatory effect of PDGF-BB. PDGF-BB increased glycosaminoglycan (GAG) chain length as shown by molecular sieve chromatography after beta-elimination to release GAG chains. Nitric oxide was the only negative regulator of biglycan as evidenced by marked downregulation in response to DETA-NO ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), a long acting nitric oxide donor and SNAP (S-nitroso-N-acetyl-l,l-penicillamine), which completely inhibited PDGF-BB-induced secretion of total [(35)S]-labeled proteoglycans and biglycan mRNA expression. Of note, the molecular weight of biglycan GAG chains was even further increased by NO donors compared to control and PDGF-BB stimulation. The current results suggest that in cardiac fibroblasts, biglycan is induced by a variety of stimuli including serum, thrombin and growth factors such as PDGF-BB and TGFbeta1. This response is counteracted by NO and enhanced by TNFalpha. Interestingly, both up- and downregulation were associated with posttranslational increase of GAG chain length.

Letter by Csont and Ferdinandy Regarding Article, “Biglycan Is Required for Adaptive Remodeling After Myocardial Infarction”

To the Editor: We read with great interest the article by Westermann et al1 in which the authors showed that biglycan expression was strongly increased in the myocardium after permanent coronary occlusion in wild-type mice. In the hearts of bgn −/ 0 mice 21 days after coronary occlusion, however, collagen matrix organization was impaired, and several parameters of left ventricular function were deteriorated. Furthermore, these animals showed increased mortality rates after infarction. The authors conclude that the complete absence of biglycan is deleterious and prevents the establishment of a stable infarct scar and hemodynamic adaptation, suggesting that biglycan is required for proper infarct healing. Although the observations of the authors are most valuable, the major conclusion of the authors1 is …

Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: consequences for bone repair during periodontal disease

A prime pathogenic agent associated with periodontitis is lipopolysaccharide (LPS) derived from Porphyromonas gingivalis. This study investigated the effects of P. gingivalis LPS on osteoblasts, which are responsible for alveolar bone repair. Bone cells were obtained from explants of rat alveolar bone chips and cultured with 0–200 ng ml−1 of P. gingivalis LPS. Porphyromonas gingivalis LPS significantly increased cell proliferation and inhibited osteoblast differentiation, as judged by reduced alkaline phosphatase activity. Analysis of biglycan mRNA and protein levels indicated that P. gingivalis LPS significantly delayed the normally high expression of biglycan during the early stages of culture, which are associated with cell proliferation and early differentiation of progenitor cells. In the presence of P. gingivalis LPS, decorin expression by the alveolar bone cells was reduced during periods of culture relating to collagen fibrillogenesis and mineral deposition. Analysis of glycosaminoglycan chains conjugated to these proteoglycans suggested that in the presence of P. gingivalis LPS, dermatan sulfate persisted within the matrix. This study suggests that P. gingivalis LPS influences the expression and processing of decorin and biglycan in the matrix, altering alveolar bone cell activity and osteoblast phenotype development. The consequences of this altered expression in relation to hindering bone repair as part of the cycle of events during periodontal disease are discussed.

Is cultured tendon fibroblast a good model to study tendon healing?

Cultured tendon fibroblasts (CTFs) from intact explants are widely used to study tendon healing in vitro. The significance of these findings may rely on similarities between CTFs and healing tendon fibroblasts in situ. Our purpose was to compare CTFs with fibroblasts cultured from healing tendons. We cultured CTFs from intact and healing tendons at day 7 and day 14 postinjury in a rat model of patellar donor site injury. The mRNA expression of COL1A1, COL3A1, decorin, and biglycan, with or without supplementation of 1 ng/mL TGF-beta1, was compared by quantitative real-time RT-PCR. The expression of proliferation cell nuclear antigen (PCNA) and alpha-smooth muscle actin (alpha-SMA) was determined by immunostain. COL3A1 and decorin mRNA in CTFs was lower as compared to day 7 healing fibroblasts, but its biglycan mRNA level was higher than day 14 healing fibroblasts. TGF-beta1 increased COL1A1 and decorin mRNA in CTFs, but decreased the mRNA of all four genes in day 7 healing tendon fibroblasts. CTFs exhibited lower PCNA immunopositivity as compared to day 7 and day 14 healing fibroblasts, but a higher alpha-SMA immunopositivity than cultured day 14 healing fibroblasts. These findings showed that CTFs did not resemble healing tendon cells with respect to major cellular activities related to tendon healing. Thus, fibroblasts from healing tendon may be a more appropriate model for studying cellular activities in tendon healing.

Differences in mRNA and protein expression of small proteoglycans in vaginal wall tissue from women with and without stress urinary incontinence

To investigate changes in mRNA and protein levels of biglycan (BGN), decorin (DCN) and fibromodulin (FMOD) in vaginal wall tissue from women with stress urinary incontinence (SUI) compared to menstrual-cycle matched continent women. We determined mRNA expressions of BGN, DCN and FMOD by quantitative real-time PCR. They were localized in vaginal wall tissue by immunohistochemistry. We performed western blot analysis to examine protein expression. BGN, DCN and FMOD co-localized with collagen and elastin in the extracellular matrix (ECM) of vaginal wall tissue from both groups. The mRNA expression of FMOD was significantly lower in cases versus controls in the proliferative phase (P = 0.03). DCN mRNA expression in cases was higher in the proliferative (P = 0.05) and secretory phases (P = 0.02) versus controls. BGN mRNA expression showed no significant differences in either phase. Protein expression of FMOD in cases was lower in the proliferative phase versus controls (six out of nine pairs), whereas DCN and BGN protein expression in the secretory phase in cases was higher (seven out of nine pairs). BGN, DCN and FMOD expressions in vaginal wall tissue differ in women with SUI and are hormonally modulated. Differences in small proteoglycans may contribute to the altered pelvic floor connective tissues found in these women.

Increased expression of aggrecan and biglycan mRNA in Achilles tendinopathy

To determine the expression of mRNA encoding the proteoglycans aggrecan, versican, biglycan and decorin in mid-tendon samples of chronic painful Achilles tendinopathy and ruptured Achilles tendons, compared with normal tendons. Total RNA isolated from frozen tendon samples (14 normal, 13 painful, 14 ruptured) was assayed by relative quantitative reverse transcription polymerase chain reaction for aggrecan, versican, biglycan and decorin mRNA, normalized using 18S rRNA. Differences between sample groups were tested by univariate analysis of variance with age as co-variate. In normal tendon samples expression of each of the proteoglycan mRNA decreased with increasing age. Decorin mRNA was the most highly-expressed of the proteoglycan mRNA, while versican mRNA expression was higher (3.8-fold) than that of aggrecan. In painful tendinopathy both aggrecan and biglycan mRNA expression increased (more than 10-fold and 5-fold, respectively) compared with normal tendon samples, but levels of versican and decorin mRNA were not significantly changed. In ruptured tendons the levels of aggrecan, biglycan and versican mRNA were not changed compared with normal tendon samples, but decorin mRNA decreased markedly. Increased aggrecan and biglycan mRNA expression in painful tendinopathy resembles the pattern in fibrocartilaginous regions of tendon, and may reflect an altered mechanical environment at the site of the lesion. Increased aggrecan mRNA expression may underlie the increase in glycosaminoglycan observed in painful tendinopathy.

Decorin and biglycan expression is differentially altered in several muscular dystrophies

Biglycan and decorin are small extracellular proteoglycans that interact with cytokines, whose activity they may modulate, and with matrix proteins, particularly collagens. To better understand their role in muscle fibrosis, we investigated expression of decorin and biglycan transcripts and protein in muscle of several forms of muscular dystrophy, and also expression of perlecan, an extracellular proteoglycan unrelated to collagen deposition. In Duchenne muscular dystrophy (DMD) and LAMA2-mutated congenital muscular dystrophy (MDC1A) we also quantitated transcript levels of the profibrotic cytokine TGF-beta1. We examined muscle biopsies from nine DMD patients, aged 2-8 years; 14 BMD (Becker muscular dystrophy) patients (nine aged 1-5 years; five aged 30-37 years); four MDC1A patients (aged 2-7 years); six dysferlin-deficient patients (aged 19-53 years) with mutation ascertained in two, and normal expression of proteins related to limb girdle muscular dystrophies in the others; 10 sarcoglycan-deficient patients: seven with alpha-sarcoglycan mutation, two with beta-sarcoglycan mutation and one with gamma-sarcoglycan mutation (five aged 8-15 years; five aged 26-43 years); and nine children (aged 1-6 years) and 12 adults (aged 16-61 years) suspected of neuromuscular disease, but who had normal muscle on biopsy. Biglycan mRNA levels varied in DMD and MDC1A depending on the quantitation method, but were upregulated in BMD, sarcoglycanopathies and dysferlinopathy. Decorin mRNA was significantly downregulated in DMD and MDC1A, whereas TGF-beta1 was significantly upregulated. Decorin mRNA was normal in paediatric BMD, but upregulated in adult BMD, sarcoglycanopathies and dysferlinopathy. Perlecan transcript levels were similar to those of age-matched controls in all disease groups. By immunohistochemistry, decorin and biglycan were mainly localized in muscle connective tissue; their presence increased in relation to increased fibrosis in all dystrophic muscle. By visual inspection, decorin bands on immunoblot did not differ from those of age-matched controls in all patient groups. However, when the intensity of the bands was quantitated against vimentin and normalized against sarcomeric actin, in DMD and MDC1A the ratio of band intensities was significantly lower than in age-matched controls. Variations in the transcript and protein levels of these proteoglycans in different muscular dystrophies probably reflect the variable disruption of extracellular matrix organization that occurs in these diseases. The significantly lowered decorin levels in DMD and MDC1A may be related to the increased TGF-beta1 levels, suggesting a therapeutic role of decorin in these severe dystrophies.

Kinetics of biglycan, decorin and thrombospondin-1 in mercuric chloride-induced renal tubulointerstitial fibrosis

We investigated the kinetics of decorin, biglycan and thrombospondin-1 in mercuric chloride-treated Brown Norway (BN) rats. BN rats were injected subcutaneously with 1 mg/kg b.w. of mercuric chloride one or three times. The kidney was examined histopathologically and the kinetics of decorin, biglycan and thrombospondin-1 was also examined using immunohistochemistry and real time RT-PCR. As a result, mercuric chloride induced tubular injury and subsequent tubulointerstitial fibrosis. In this lesion, the expression of thrombospondin-1 mRNA was most prominently elevated. The expression of decorin mRNA was next, but biglycan mRNA expression was not elevated. Moreover, decorin and thrombospondin-1 proteins were localized in tubular epithelial cells and peritubular interstitium. Moreover, kinetics of their mRNA expressions was relatively similar to the kinetics of TGF-β1 mRNA expression previously reported. The present findings suggest that decorin and thrombospondin-1 may participate in the development of tubulointerstitial fibrosis and may have some relation with TGF-β1 in mercuric chloride-treated BN rats.

Transforming Growth Factor-β (TGF-β) Type I Receptor/ALK5-dependent Activation of the GADD45β Gene Mediates the Induction of Biglycan Expression by TGF-β

We have recently shown that induction of biglycan (BGN) expression by transforming growth factor-beta1 (TGF-beta1) required sequential activation of both Smad and p38 mitogen-activated protein kinase signaling (Ungefroren, H., Lenschow, W., Chen, W.-B., and Kalthoff, H. (2003) J. Biol. Chem. 278, 11041-11049). Here, we have analyzed the receptors through which TGF-beta1 controls expression of BGN and GADD45beta, the latter of which is postulated to link early Smad signaling to delayed activation of p38. Ectopic expression of a dominant-negative mutant of the TGF-beta type II receptor in PANC-1 cells abrogated TGF-beta-induced BGN up-regulation. Similarly, inhibition of the TGF-beta type I receptor/ALK5 with either SB431542 or by enforced stable expression of a kinase-dead mutant greatly attenuated the TGF-beta effect on both BGN and GADD45beta expression in PANC-1 and MG-63 cells. The enhancing effect of ALK5 on TGF-beta-mediated GADD45beta and BGN expression and on GADD45beta promoter activity was also dependent on its ability to activate Smad signaling, because an ALK5 mutant defective in Smad activation (TbetaRImL45) but with an otherwise functional kinase domain failed to mediate these responses. The TGF-beta/ALK5 effect on p38 activation and BGN expression was mimicked by overexpression of GADD45beta alone (in the absence of TGF-beta stimulation) and suppressed upon antisense inhibition of GADD45beta expression. These results show that TGF-beta induces BGN expression through (the Smad-activating function of) ALK5 and GADD45beta and suggest that the sensitivity of MyD118 to activation by TGF-beta, which varies between tissues, ultimately determines the strength of the TGF-beta effect on BGN.