COL3A1, COL6A3, and SERPINH1 Are Related to Glucocorticoid-Induced Osteoporosis Occurrence According to Integrated Bioinformatics Analysis.

BACKGROUND Glucocorticoid-induced osteoporosis (GIOP) represents the most frequently seen type of secondary osteoporosis, a systemic skeleton disorder. Numerous factors are associated with GIOP occurrence, but there are no specific diagnostic and therapeutic biomarkers for GIOP so far. MATERIAL AND METHODS In this work, gene modules related to GIOP were screened through weighted gene coexpression network analysis. Moreover, protein-protein interaction (PPI) networks and gene set enrichment analysis (GSEA) were carried out for hub genes. In addition, microarray GSE30159 dataset was used as a training set to analyze gene expression within bone biopsy samples from patients with endogenous Cushing's syndrome with GIOP and from normal controls. GSE129228 was used as the test set for investigating the hub gene involvement within GIOP. RESULTS According to our results, the turquoise module showed clinical significance, and 10 genes (COL3A1, POSTN, COL6A3, COL14A1, SERPINH1, ASPN, OGN, THY1, NID2, and TNMD) were discovered to be the "real" hub genes within coexpression as well as PPI networks. GSEA showed that the interaction of extracellular matrix receptors together with the focal adhesion pathway had significant enrichment within samples with high COL3A1 and COL6A3 expression. After the results from both test and training sets were overlapped, SERPINH1 was also significantly altered between GIOP and normal control samples. CONCLUSIONS COL3A1, COL6A3, and SERPINH1 were identified to be the candidate biomarkers for GIOP.

A new single nucleotide polymorphism affects the predisposition to thoracic ossification of the posterior longitudinal ligament.

BACKGROUND: Thoracic ossification of the posterior longitudinal ligament (T-OPLL) is one of the common factors that cause thoracic spinal stenosis, which results in intractable myelopathy and radiculopathy. Our previous study first reported rs201153092A site mutation in the collagen 6A1 (COL6A1) gene as a potentially pathogenic locus for T-OPLL. We aimed to determine whether the rs201153092A site mutation causes abnormal expression of the COL6A1 in Han Chinese patients with T-OPLL and whether this locus is also associated with cervical-OPLL. METHODS: Peripheral blood was collected from a total of 60 patients with T-OPLL disease (30 patients carrying the rs201153092A site mutation in COL6A1 and 30 wild-type patients) and 400 northern Chinese individuals (200 cervical-OPLL patients and 200 control subjects) using the Sequenom system. The expression of COL6A1 was analyzed by enzyme-linked immunosorbent assay, reverse transcription-quantitative polymerase chain reaction, and Western blotting. RESULTS: rs201153092A mutation resulted in markedly increased COL6A1 gene expression levels in peripheral blood samples. The allele frequency and genotype frequency results showed that this locus is no difference between cervical-OPLL patients and controls. CONCLUSIONS: The rs201153092A site mutation of COL6A1 can significantly increase the expression of COL6A1. The COL6A1 gene rs201153092A site polymorphism is a potential pathogenic mutation in T-OPLL disease, which may be only associated with the occurrence of T-OPLL.

Fra-2-expressing macrophages promote lung fibrosis in mice.

Idiopathic Pulmonary Fibrosis (IPF) is a deadly disease with limited therapies. Tissue fibrosis is associated with Type 2 immune response, although the causal contribution of immune cells is not defined. The AP-1 transcription factor Fra-2 is upregulated in IPF lung sections and Fra-2 transgenic mice (Fra-2tg) exhibit spontaneous lung fibrosis. Here we show that Bleomycin-induced lung fibrosis is attenuated upon myeloid-inactivation of Fra-2 and aggravated in Fra-2tg bone marrow chimeras. Type VI collagen (ColVI), a Fra-2 transcriptional target, is up-regulated in three lung fibrosis models, and macrophages promote myofibroblast activation in vitro in a ColVI- and Fra-2-dependent manner. Fra-2 or ColVI inactivation does not affect macrophage recruitment and alternative activation, suggesting that Fra-2/ColVI specifically controls the paracrine pro-fibrotic activity of macrophages. Importantly, ColVI knock-out mice (KO) and ColVI-KO bone marrow chimeras are protected from Bleomycin-induced lung fibrosis. Therapeutic administration of a Fra-2/AP-1 inhibitor reduces ColVI expression and ameliorates fibrosis in Fra-2tg mice and in the Bleomycin model. Finally, Fra-2 and ColVI positively correlate in IPF patient samples and co-localize in lung macrophages. Therefore, the Fra-2/ColVI pro-fibrotic axis is a promising biomarker and therapeutic target for lung fibrosis, and possibly other fibrotic diseases.

Collagen Type VI Alpha 3 Chain Promotes Epithelial-Mesenchymal Transition in Bladder Cancer Cells via Transforming Growth Factor ß (TGF-ß)/Smad Pathway.

BACKGROUND Collagen type VI alpha 3 chain (COL6A3) has been proven to be a biomarker in the occurrence and development of bladder cancer, which is the most common malignant tumor in the urinary system. This study aimed to explore the effect and molecular mechanism of COL6A3 on EMT in vitro induced by TGF-ß/Smad in bladder carcinoma. MATERIAL AND METHODS There were 42 patients included in the Kaplan-Meier survival analysis. A cell counting kit-8 (CCK-8) assay and an angiogenesis assay were used to measure cell proliferation and tube formation, respectively. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qPCR) were conducted for the proteins and mRNAs expression. RESULTS COL6A3 was highly expressed in tissues and cells of bladder cancer. COL6A3 silencing could inhibit the cell proliferation and angiopoiesis. In addition, COL6A3 silencing obviously suppressed the levels of matrix metalloproteinase-2 (MMP2), Matrix metalloproteinase-9 (MMP9), and vimentin. On the contrary, the levels of epithelium-specific cell-cell adhesion molecule (E-cadherin) and tumor inhibitor of metalloproteinase-1 (TIMP-1) were significantly increased. Furthermore, we found that COL6A3 silencing reduced the activity of p-Smad2, p-Smad3, and transforming growth factor ß (TGF-ß). CONCLUSIONS COL6A3 could influence the viability and angiogenesis of bladder cancer cells. COL6A3 may have a certain relationship with the TGF-ß/Smad-induced EMT process.

Silencing of COL1A2, COL6A3, and THBS2 inhibits gastric cancer cell proliferation, migration, and invasion while promoting apoptosis through the PI3k-Akt signaling pathway.

This study explores the effect of COL1A2, COL6A3, and THBS2 gene silencing on proliferation, migration, invasion, and apoptosis of gastric cancer cells through the PI3K-Akt signaling pathway. The gastric cancer microarray expression data (GSE19826, GSE79973, and GSE65801) was analyzed. Gastric cancer tissues and corresponding adjacent normal tissues were extracted from patients. Positive expression rate of PI3K, Akt, and p-Akt was measured with immunohistochemistry. Two cell lines, BGC-823 and SGC-7901, were transfected and cells were grouped into blank, negative control, COL1A2-shRNA, COL6A3-shRNA, and THBS2-shRNA groups. Expressions of COL1A2, COL6A3, and THBS2 in gastric cancer cells transfected with corresponding silencing sequences were evaluated by RT-qPCR and Western blot. MTT assay, Transwell, and cell scratch tests were conducted to evaluate cell proliferation, invasion, and migration capacity, respectively. Flow cytometry was used to evaluate cell cycle distribution and apoptosis. The positive expression of PI3K, Akt, and p-Akt was higher in gastric cancer tissues compared with adjacent normal tissues, and the mRNA expression of COL1A2, COL6A3, and THBS2 was increased in gastric cancer tissues. Akt, p-Akt, and PI3K expression drastically decreased in cells transfected with COL1A2, COL6A3, and THBS2 silencing sequences. Cells transfected with COL1A2, COL6A3, and THBS2 silencing sequences exhibited promoted apoptosis but inhibited proliferation, migration, and invasion. This study demonstrates that COL1A2, COL6A3, and THBS2 gene silencing inhibits gastric cancer cell proliferation, migration, and invasion while promoting apoptosis through the PI3K-Akt signaling pathway.

Collagen VI and hyaluronan: the common role in breast cancer.

Collagen VI and hyaluronan are widely distributed extracellular matrix macromolecules that play a crucial role in tissue development and are highly expressed in cancers. Both hyaluronan and collagen VI are upregulated in breast cancer, generating a microenvironment that promotes tumour progression and metastasis. A growing number of studies show that these two molecules are involved in inflammation and angiogenesis by recruiting macrophages and endothelial cells, respectively. Additionally, collagen VI induces epithelial-mesenchymal transition that is correlated to increased synthesis of hyaluronan in mammary cells. Hyaluronan has also a specific role in cellular functions that depends mainly on the size of the polymer, whereas the effect of collagen VI in tumour progression may be the result of the intact molecule or the C5 peptide of α3(VI) chain, known as endotrophin. Collectively, these findings strongly support the parallel role of these molecules in tumour progression and suggest that they may be used as prognostic factors for the breast cancer treatment.

Transcriptome analysis reveals differential splicing events in IPF lung tissue.

Idiopathic pulmonary fibrosis (IPF) is a complex disease in which a multitude of proteins and networks are disrupted. Interrogation of the transcriptome through RNA sequencing (RNA-Seq) enables the determination of genes whose differential expression is most significant in IPF, as well as the detection of alternative splicing events which are not easily observed with traditional microarray experiments. We sequenced messenger RNA from 8 IPF lung samples and 7 healthy controls on an Illumina HiSeq 2000, and found evidence for substantial differential gene expression and differential splicing. 873 genes were differentially expressed in IPF (FDR<5%), and 440 unique genes had significant differential splicing events in at least one exonic region (FDR<5%). We used qPCR to validate the differential exon usage in the second and third most significant exonic regions, in the genes COL6A3 (RNA-Seq adjusted pval = 7.18e-10) and POSTN (RNA-Seq adjusted pval = 2.06e-09), which encode the extracellular matrix proteins collagen alpha-3(VI) and periostin. The increased gene-level expression of periostin has been associated with IPF and its clinical progression, but its differential splicing has not been studied in the context of this disease. Our results suggest that alternative splicing of these and other genes may be involved in the pathogenesis of IPF. We have developed an interactive web application which allows users to explore the results of our RNA-Seq experiment, as well as those of two previously published microarray experiments, and we hope that this will serve as a resource for future investigations of gene regulation in IPF.

G-CSF prevents progression of diabetic nephropathy in rat.

BACKGROUND: The protective effects of granulocyte colony-stimulating factor (G-CSF) have been demonstrated in a variety of renal disease models. However, the influence of G-CSF on diabetic nephropathy (DN) remains to be examined. In this study, we investigated the effect of G-CSF on DN and its possible mechanisms in a rat model. METHODS: Otsuka Long-Evans Tokushima Fatty (OLETF) rats with early DN were administered G-CSF or saline intraperitoneally. Urine albumin creatinine ratio (UACR), creatinine clearance, mesangial matrix expansion, glomerular basement membrane (GBM) thickness, and podocyte foot process width (FPW) were measured. The levels of interleukin (IL)-1ß, transforming growth factor (TGF)-ß1, and type IV collagen genes expression in kidney tissue were also evaluated. To elucidate the mechanisms underlying G-CSF effects, we also assessed the expression of G-CSF receptor (G-CSFR) in glomeruli as well as mobilization of bone marrow (BM) cells to glomeruli using sex-mismatched BM transplantation. RESULTS: After four weeks of treatment, UACR was lower in the G-CSF treatment group than in the saline group (p<0.05), as were mesangial matrix expansion, GBM thickness, and FPW (p<0.05). In addition, the expression of TGF-ß1 and type IV collagen and IL-1ß levels was lower in the G-CSF treatment group (p<0.05). G-CSFR was not present in glomerular cells, and G-CSF treatment increased the number of BM-derived cells in glomeruli (p<0.05). CONCLUSIONS: G-CSF can prevent the progression of DN in OLETF rats and its effects may be due to mobilization of BM cells rather than being a direct effect.

Adipose tissue collagen and inflammation in nonobese Asian Indian men.

OBJECTIVE: Obesity is thought to be the driving force for activation of adipose tissue (AT) collagen production and inflammation as well as systemic insulin resistance. The objective of this study was to determine whether these AT abnormalities can be found independent of obesity in the presence of systemic insulin resistance. RESEARCH DESIGN AND METHODS: Thirty-eight normoglycemic men (14 Asian Indians and 24 white) were enrolled in the study and matched for age, body mass index, and total body fat. Subjects underwent anthropometric measurement, total body fat determination by underwater weighing, euglycemic-hyperinsulinemic clamps, and abdominal sc AT biopsy for mRNA extraction and gene expression determination. Fasting blood was collected for adipokine measurements. RESULTS: Both groups were matched for age, body mass index, and percentage of total body fat. Subcutaneous abdominal AT mRNA expression was significantly higher for Col6a3 as well as genes associated with inflammation, CD68, MAC1, and MCP1 in Asian Indians compared with whites. Asian Indian men had significantly lower rates of glucose disposal and lower plasma adiponectin concentration. Plasma high-sensitivity C-reactive protein levels showed a trend towards higher levels in Asian Indian men. CONCLUSIONS: Increased col6a3 and macrophage infiltration in AT along with increased systemic insulin resistance is present independent of body fat content in young Asian Indian men, thus suggesting that AT dysfunction associates with systemic insulin resistance regardless of AT mass.

Identification of the up-regulation of TP-alpha, collagen alpha-1(VI) chain, and S100A9 in esophageal squamous cell carcinoma by a proteomic method.

Esophageal squamous cell carcinoma (ESCC) is one of the most common primary malignant tumor of digestive tract. However, the early diagnosis and molecular mechanisms that underlie tumor formation and progression have been progressed less. To identify new biomarkers for ESCC, we performed a comparative proteomic research. Isobaric tags for relative and absolute quantitation-based proteomic method was used to screen biomarkers between ESCC and normal. 802 non-redundant proteins were identified, 39 of which were differentially expressed with 1.5-fold difference (29 up-regulated and 10 down-regulated). Through Swiss-Prot and GO database, the location and function of differential proteins were analyzed, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc. Among the differentially expressed proteins, TP-alpha, collagen alpha-1(VI) chain and S100A9 were verified to be upregulated in 77.19%, 75.44% and 59.65% of ESCC by immunohistochemistry and western-blot. Diagnostic value of these three proteins was validated. These results provide new insights into ESCC biology and potential diagnostic and therapeutic biomarkers, which suggest that TP-alpha, collagen alpha-1(VI) chain and S100A9 are potential biomarkers of ESCC, and may play an important role in tumorigenesis and development of ESCC.

Macrophages: a minimally invasive tool for monitoring collagen VI myopathies.

INTRODUCTION: Collagen VI expression was tested in peripheral blood macrophages from patients with collagen VI-related myopathies and compared with muscle biopsy. METHODS: RNA and protein studies were performed in blood macrophages from 5 patients previously diagnosed with either Ullrich congenital muscular dystrophy (UCMD) or Bethlem myopathy (BM). The full spectrum of possible genotypes was considered, including both dominant and recessive UCMD and BM cases. RESULTS: In the dominant BM patient, no collagen VI alterations were detectable in macrophages or muscle biopsy. In the remaining patients, the protein defect caused by the selected mutations, as well as the transcriptional abnormalities, were readily detectable in macrophages, at levels comparable to those observed in muscle biopsy samples and cultured skin fibroblasts. CONCLUSIONS: Our data support the suitability of peripheral blood macrophages as a reliable, minimally invasive tool for supplementing or replacing muscle/skin biopsies in the diagnosis and monitoring of collagen VI-related myopathies.

Adipose tissue collagen VI in obesity.

OBJECTIVES: Basic science studies show that the extracellular matrix of adipose tissue, mainly represented by collagen VI, is dysfunctional in obesity and contributes to the development of the metabolic syndrome. We hypothesized in humans that increased collagen VI alpha3-subunit (COL6A3) mRNA is associated with adipose tissue macrophage chemotaxis and inflammation and that weight gain is accompanied by changes in the expression of COL6A3. RESEARCH DESIGN AND METHODS: Adipose tissue biopsies were obtained from a cross-sectional study (n = 109), an overfeeding study (n = 9), and a pioglitazone treatment study (n = 14). Adipose tissue gene expression was measured by quantitative RT-PCR, immunohistochemistry, and adipocyte sizing by fixation with osmium and Coulter counting. Body composition was measured by dual-energy x-ray absorptiometry and visceral adipose tissue by computed tomography. Patients with high or low COL6A3 mRNA were compared by one-way ANOVA. RESULTS: In humans, immunohistochemistry revealed that COL6 is present in adipose tissue extracellular matrix. COL6A3 mRNA is correlated with body mass index (r = 0.60, P < 0.0001) and fat mass (r = 0.41, P < 0.0001). COL6A3 expression was similar in obese vs. type 2 diabetes patients. Obese subjects with high COL6A3 mRNA had greater visceral adipose tissue mass (P < 0.05), lower size of small and medium adipocytes (P < 0.05), more CD68+ and CD163/MAC2+ macrophages, and increased macrophage inflammatory protein-1alpha and macrophage chemoattractant protein-1alpha mRNA (P < 0.05). Eight weeks of overfeeding increased body weight and COL6A3 mRNA (P < 0.05). Pioglitazone decreased COL6A3 mRNA, and the change was inversely proportional to baseline COL6A3 mRNA (r = -0.95, P < 0.0001). CONCLUSION: These results are consistent with basic science data, suggesting that COL6A3 might contribute to adipose tissue inflammation.

Advanced osteoarthritis in humans is associated with altered collagen VI expression and upregulation of ER-stress markers Grp78 and bag-1.

To test the hypothesis that a perturbation of endoplasmic reticulum (ER) function is involved in the pathogenesis of osteoarthritis (OA), articular cartilage was isolated from non-OA patients secondary to resection of osteo- or chondrosarcomas. Intra-joint samples of minimal and advanced osteoarthritic cartilage were isolated from patients undergoing total knee arthroplasty and scored for disease severity. Glucose-regulated protein-78 (grp78) and bcl-2-associated athanogene-1 (bag-1) were detected via immunofluorescence as markers of non-homeostatic ER function. Additionally, the expression of type VI collagen and its integrin receptor, NG2, was determined to examine cartilage matrix health and turnover. There was an upregulation of grp78 in advanced OA, and variable expression in minimal OA. Non-OA cartilage was consistently grp78 negative. The downstream regulator bag-1 was also upregulated in OA compared with normal cartilage. Collagen VI was mainly cell-associated in non-OA cartilage, with a more widespread distribution observed in OA cartilage along with increased intracellular staining intensity. The collagen VI integral membrane proteoglycan receptor NG2 was downregulated in advanced OA compared with its patient-matched minimally involved cartilage sample. These results suggest that chondrocytes exhibit ER stress during OA, in association with upregulation of a large secreted molecule, type VI collagen.

Detection and characterization of chondroid metaplasia in canine atrioventricular valves.

The atrioventricular valves of 25 dogs of different breeds and age were examined grossly and microscopically following histochemical staining and immunohistochemical labelling for collagen types I, III and VI, and for fibronectin and laminin. Foci of cartilage were identified in the tricuspid septal leaflet within the fibrosa (n=21) or spongiosa (n=3). These were further characterized as either fibrocartilage, predominantly composed of collagens I and VI, or hyaline cartilage consisting of laminin and collagens III and VI. Eighteen of the dogs were of large breed and seven of small breed. Retrospective echocardiographic findings were available from five cases and in three of these a hyperechogenic structure was identified corresponding to the cartilage focus (0.1, 1.12 and 5.63 mm(2) in size). The clinical significance and mechanism of formation of these cartilaginous foci remain undetermined, although factors such as breed, size and concurrent chronic valvular disease may be significant.

Production of type VI collagen by human macrophages: a new dimension in macrophage functional heterogeneity.

Macrophages derived from human blood monocytes perform many tasks related to tissue injury and repair. The main effect of macrophages on the extracellular matrix is considered to be destructive in nature, because macrophages secrete metalloproteinases and ingest foreign material as part of the remodeling process that occurs in wound healing and other pathological conditions. However, macrophages also contribute to the extracellular matrix and hence to tissue stabilization both indirectly, by inducing other cells to proliferate and to release matrix components, and directly, by secreting components of the extracellular matrix such as fibronectin and type VIII collagen, as we have recently shown. We now report that monocytes and macrophages express virtually all known collagen and collagen-related mRNAs. Furthermore, macrophages secrete type VI collagen protein abundantly, depending upon their mode of activation, stage of differentiation, and cell density. The primary function of type VI collagen secreted by macrophages appears to be modulation of cell-cell and cell-matrix interactions. We suggest that the production of type VI collagen is a marker for a nondestructive, matrix-conserving macrophage phenotype that could profoundly influence physiological and pathophysiological conditions in vivo.

Use of a cDNA microarray to determine molecular mechanisms involved in grey platelet syndrome.

The grey platelet syndrome (GPS) is a bleeding disorder of unknown aetiology with phenotypic and genetic heterogeneity. Affected patients exhibit macrothrombocytopenia, decreased alpha-granule content and, sometimes, myelofibrosis. We used microarray technology to investigate changes in gene expression that might reveal mechanisms involved in GPS. The expression of 4900 unique genes and expressed sequence tags was evaluated in fibroblasts from a GPS patient; normal fibroblasts provided the reference standard. Genes that were differentially regulated in the GPS cells were categorized into gene clusters based upon similarity/differences of expression differences. The results showed that genes with functional similarities clustered together. This analysis revealed significant upregulation of selected biological processes involving the production of cytoskeleton proteins, including fibronectin 1, thrombospondins 1 and 2, and collagen VI alpha. These genes appear to play a role in the pathogenesis of GPS. Indeed, Northern blot analyses confirmed that fibronectin, thrombospondin and matrix metalloprotease-2 were overexpressed in GPS fibroblasts compared with normal fibroblasts. Moreover, immunohistochemistry studies revealed robust fibronectin staining in GPS fibroblasts compared with normal ones. Our findings support the feasibility of using cDNA microarray techniques to detect distinctive and informative differences in gene expression patterns relevant to GPS, and suggest that the molecular basis for myelofibrosis in GPS involves upregulation of cytoskeleton proteins.

Remodeling of the extracellular matrix through overexpression of collagen VI contributes to cisplatin resistance in ovarian cancer cells.

The mechanisms of drug resistance in cancer are poorly understood. Serial analysis of gene expression (SAGE) profiling of cisplatin-resistant and sensitive cells revealed many differentially expressed genes. Remarkably, many ECM genes were elevated in cisplatin-resistant cells. COL6A3 was one of the most highly upregulated genes, and cultivation of cisplatin-sensitive cells in the presence of collagen VI protein promoted resistance in vitro. Staining of ovarian tumors with collagen VI antibodies confirmed collagen VI expression in vivo and suggested reorganization of the extracellular matrix in the vicinity of the tumor. Furthermore, the presence of collagen VI correlated with tumor grade, an ovarian cancer prognostic factor. These results suggest that tumor cells may directly remodel their microenvironment to increase their survival in the presence of chemotherapeutic drugs.

Biosynthesis of type VI collagen by glioblastoma cells and possible function in cell invasion of three-dimensional matrices.

The biosynthesis of type VI collagen was studied in human glioblastoma cell line, U-87 MG. The effects of ascorbic acid on type VI collagen synthesis and secretion were investigated. After ascorbic acid treatment, type VI collagen in cell layers increased from 4.48% in control to 6.63% in the ascorbic acid treated cultures, an increase of 48%. The effect of ascorbic acid on type VI collagen synthesized by glioblastoma cells was lower than that reported for osteosarcoma cells (Engvall et al., 1986). The reason for these differences is still under investigation. The function of type VI collagen in glioblastoma cells is still unknown. We utilized the collagen gel system to elucidate the possible roles of type VI collagen in glioblastoma cells in vitro. Glioblastoma cells in collagen gels showed a stellate shape with long, branched processes in all directions. The strong positive reactivity of type VI collagen detected on cell bodies and cell processes by anti-type VI collagen antibody indicated that this specific collagen was associated with cell surfaces and processes, without releasing or diffusing into the gels. Type VI collagen was directly involved in the cell process extension. When living cells were treated with anti-type VI collagen antibody, a variation of cell morphology was observed. Instead of a stellate shape with processes, cells formed clusters without or with very short processes. These data suggest that type VI collagen, synthesized and secreted by glioblastoma cells, may play a role in tumor cell adhesion and spreading, and enhance cell process extension, penetration, and invasion into collagen gels.