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The Ehlers-Danlos Syndrome (EDS) is a heritable connective tissue disorder characterized by hyperextensible skin, joint hypermobility and soft tissue fragility. The classic subtype of EDS is caused by mutations in one of the type V collagen genes (COL5A1 and COL5A2). Most mutations affect the type V collagen helical domain and lead to a diminished or structurally abnormal type V collagen protein. Remarkably, only two mutations were reported to affect the extended, highly conserved N-propeptide domain, which plays an important role in the regulation of the heterotypic collagen fibril diameter. We identified a novel COL5A1 N-propeptide mutation, resulting in an unusual but severe classic EDS phenotype and a remarkable splicing outcome. We identified a novel COL5A1 N-propeptide acceptor-splice site mutation (IVS6-2A>G, NM_000093.3_c.925-2A>G) in a patient with cutaneous features of EDS, severe progressive scoliosis and eye involvement. Two mutant transcripts were identified, one with an exon 7 skip and one in which exon 7 and the upstream exon 6 are deleted. Both transcripts are expressed and secreted into the extracellular matrix, where they can participate in and perturb collagen fibrillogenesis, as illustrated by the presence of dermal collagen cauliflowers. Determination of the order of intron removal and computational analysis showed that simultaneous skipping of exons 6 and 7 is due to the combined effect of delayed splicing of intron 7, altered pre-mRNA secondary structure, low splice site strength and possibly disturbed binding of splicing factors. We report a novel COL5A1 N-propeptide acceptor-splice site mutation in intron 6, which not only affects splicing of the adjacent exon 7, but also causes a splicing error of the upstream exon 6. Our findings add further insights into the COL5A1 splicing order and show for the first time that a single COL5A1 acceptor-splice site mutation can perturb splicing of the upstream exon.

The epidermal basement membrane deteriorates with aging. We previously reported that basement membrane reconstruction not only serves to maintain epidermal stem/progenitor cells in the epidermis, but also increases collagen fibrils in the papillary dermis. Here, we investigated the mechanism of the latter action. Collagen fibrils in the papillary dermis were increased in organotypic human skin culture treated with matrix metalloproteinase and heparinase inhibitors. The expression levels of COL5A1 and COL1A1 genes (encoding collagen type V α 1 chain and collagen type I α 1 chain, respectively) were increased in fibroblasts cultured with conditioned medium from a skin equivalent model cultured with the inhibitors and in keratinocytes cultured on laminin-511 E8 fragment-coated plates. We then examined cytokine expression, and found that the inhibitors increased the expression of PDGF-BB (platelet-derived growth factor consisting of two B subunits) in epidermis. Expression of COL5A1 and COL1A1 genes was increased in cultured fibroblasts stimulated with PDGF-BB. Further, the bifunctional inhibitor hydroxyethyl imidazolidinone (HEI) increased skin elasticity and the thickness of the papillary dermis in the skin equivalent. Taken together, our data suggests that reconstructing the basement membrane promotes secretion of PDGF-BB by epidermal keratinocytes, leading to increased collagen expression at the papillary dermis.

Triple-negative breast cancer (TNBC) is an exceptionally aggressive subtype of breast cancer. Despite the recognized interplay between tumors and tumor-associated macrophages in fostering drug resistance and disease progression, the precise mechanisms leading these interactions remain elusive. Our study revealed that the upregulation of collagen type V alpha 1 (COL5A1) in TNBC tissues, particularly in chemoresistant samples, was closely linked to an unfavorable prognosis. Functional assays unequivocally demonstrated that COL5A1 played a pivotal role in fueling cancer growth, metastasis, and resistance to doxorubicin, both in vitro and in vivo. Furthermore, we found that the cytokine IL-6, produced by COL5A1-overexpressing TNBC cells actively promoted M2 macrophage polarization. In turn, TGFβ from M2 macrophages drived TNBC doxorubicin resistance through the TGFβ/Smad3/COL5A1 signaling pathway, establishing a feedback loop between TNBC cells and macrophages. Mechanistically, COL5A1 interacted with TGM2, inhibiting its K48-linked ubiquitination-mediated degradation, thereby enhancing chemoresistance and increasing IL-6 secretion. In summary, our findings underscored the significant contribution of COL5A1 upregulation to TNBC progression and chemoresistance, highlighting its potential as a diagnostic and therapeutic biomarker for TNBC.

Pericellular α3(V) collagen can affect the functioning of cells, such as adipocytes and pancreatic β cells. Here we show that α3(V) chains are an abundant product of normal mammary gland basal cells, and that α3(V) ablation in a mouse mammary tumour model inhibits mammary tumour progression by reducing the proliferative potential of tumour cells. These effects are shown to be primarily cell autonomous, from loss of α3(V) chains normally produced by tumour cells, in which they affect growth by enhancing the ability of cell surface proteoglycan glypican-1 to act as a co-receptor for FGF2. Thus, a mechanism is presented for microenvironmental influence on tumour growth. α3(V) chains are produced in both basal-like and luminal human breast tumours, and its expression levels are tightly coupled with those of glypican-1 across breast cancer types. Evidence indicates α3(V) chains as potential targets for inhibiting tumour growth and as markers of oncogenic transformation. Collagen has a role in cancer and is particularly important for breast cancer. Here the authors show that the expression of α3 type V collagen and one of its receptors- glipican-1- in the same cell, contributes to a deregulated growth of breast cancer cells.

The replacement of normal endometrial epithelium by fibrotic tissue is the pathological feature of intrauterine adhesion (IUA), which is caused by trauma to the basal layer of the endometrium. COL5A2 is a molecular subtype of collagen V that regulates collagen production in fibrotic tissue. Here, we investigated the roles of Foxf2 and Smad6 in regulating the transcription of COL5A2 and their involvement in the pathogenesis of IUA. Small interference‐mediated Foxf2 (si‐Foxf2) silencing and pcDNA3.1‐mediated Smad6 (pcDNA3.1‐Smad6) up‐regulation were performed in a TGF‐β1‐induced human endometrial stromal cell line (HESC) fibrosis model. Assessment of collagen expression by Western blotting, immunofluorescence and qRT‐PCR showed that COL5A2, COL1A1 and FN were significantly down‐regulated in response to si‐Foxf2 and pcDNA3.1‐Smad6. Transfection of lentivirus vector‐Foxf2 (LV‐Foxf2) and pcDNA3.1‐Smad6 into HESCs and qRT‐PCR showed that Foxf2 promoted COL5A2 expression and Smad6 inhibited Foxf2‐induced COL5A2 expression. Co‐immunoprecipitation, chromatin immunoprecipitation and dual‐luciferase reporter assays to detect the interaction between Foxf2 and Smad6 and their role in COL5A2 transcription showed that Foxf2 interacted with Smad6 and bond the same promoter region of COL5A2. In a rat IUA model, injection of ADV2‐Foxf2‐1810 and ADV4‐Smad6 into the uterine wall showed that Foxf2 down‐regulation and Smad6 up‐regulation decreased fibrosis and the expression of COL5A2 and COL1A1, as detected by haematoxylin/eosin, Masson trichrome staining and immunohistochemistry. Taken together, these results suggested that Foxf2 interacted with Smad6 and co‐regulated COL5A2 transcription in the pathogenesis of IUA, whereas they played opposite roles in fibrosis.

Collagen V is a key matrix protein involved in fibril nucleation and lateral fibril growth during extracellular matrix assembly. Genetic mouse models have been used to investigate the role of collagen V in tendon, which showed deficient mechanical properties and aberrant fibril structure in the absence of collagen V. However, the lasting effects of collagen V deficiency later into adulthood remain unknown, as well as the role of collagen V in maintaining a mature matrix. This study therefore investigated the long-term effects of collagen V reduction on tendon as well as its role in mature tendon matrix in adulthood. Tendon-targeted conditional Col5a1 knockout, which excises Col5a1 alleles early in development, had long-term impact on tendon structure and function in 300-day old mice. Gene expression was altered with differential expression of primarily matrix and matrix remodeling genes. Regional changes in cellular shape and density were consistent with typical behavior in tendinopathy. Fibril diameters were increased due to dysregulated lateral growth. Deficits in mechanical properties indicate a weaker tendon matrix after knockout, although deformation patterns of collagen fibrils were not affected. In contrast, inducing collagen V knockdown in a mature tendon matrix at 120-days old did not cause substantial changes in any of the above-mentioned properties in 300-day old mice. In conclusion, these findings highlight the important function of collagen V in matrix assembly that has lasting effects into later ages, even though collagen V has little role in homeostatic maintenance of a mature tendon matrix.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by progressive scarring and matrix deposition. Recent reports highlight an autoimmune component in IPF pathogenesis. We have reported anti-col(V) immunity in IPF patients. The objective of our study was to determine the specificity of col(V) expression profile and anti-col(V) immunity relative to col(I) in clinical IPF and the efficacy of nebulized col(V) in pre-clinical IPF models. Col(V) and col(I) expression profile was analyzed in normal human and IPF tissues. C57-BL6 mice were intratracheally instilled with bleomycin (0.025 U) followed by col(V) nebulization at pre-/post-fibrotic stage and analyzed for systemic and local responses. Compared to normal lungs, IPF lungs had higher protein and transcript expression of the alpha 1 chain of col(V) and col(I). Systemic anti-col(V) antibody concentrations, but not of anti-col(I), were higher in IPF patients. Nebulized col(V), but not col(I), prevented bleomycin-induced fibrosis, collagen deposition, and myofibroblast differentiation. Col(V) treatment suppressed systemic levels of anti-col(V) antibodies, IL-6 and TNF-α; and local Il-17a transcripts. Compared to controls, nebulized col(V)-induced tolerance abrogated antigen-specific proliferation in mediastinal lymphocytes and production of IL-17A, IL-6, TNF-α and IFN-γ. In a clinically relevant established fibrosis model, nebulized col(V) decreased collagen deposition. mRNA array revealed downregulation of genes specific to fibrosis (Tgf-β, Il-1β, Pdgfb), matrix (Acta2, Col1a2, Col3a1, Lox, Itgb1/6, Itga2/3) and members of the TGF-β superfamily (Tgfbr1/2, Smad2/3, Ltbp1, Serpine1, Nfkb/Sp1/Cebpb). Anti-col(V) immunity is pathogenic in IPF, and col(V)-induced tolerance abrogates bleomycin-induced fibrogenesis and down regulates TGF- β-related signaling pathways.

Genetic associations for keratoconus could be useful for understanding disease pathogenesis and discovering biomarkers for early detection of the disease. We conducted a systematic review and meta-analysis to summarize all reported genetic associations for the disease. We searched in the MEDLINE, Embase, Web of Science, and HuGENET databases for genetic studies of keratoconus published from 1950 to June 2016. The summary odds ratio and 95% confidence intervals of all polymorphisms were estimated using the random-effect model. Among 639 reports that were retrieved, 24 fulfilled required criteria as eligible studies for meta-analysis, involving a total of 53 polymorphisms in 28 genes/loci. Results of our meta-analysis lead to the prioritization of 8 single-nucleotide polymorphisms (SNPs) in 6 genes/loci for keratoconus in Whites. Of them 5 genes/loci were originally detected in genome-wide association studies, including FOXO1 (rs2721051, P = 5.6 × 10 −11 ), RXRA-COL5A1 (rs1536482, P = 2.5 × 10 −9 ), FNDC3B (rs4894535, P = 1.4 × 10 −8 ), IMMP2L (rs757219, P = 6.1 × 10 −7 ; rs214884, P = 2.3 × 10 −5 ), and BANP-ZNF469 (rs9938149, P = 1.3 × 10 −5 ). The gene COL4A4 (rs2229813, P = 1.3 × 10 −12 ; rs2228557, P = 4.5 × 10 −7 ) was identified in previous candidate gene studies. We also found SNPs in 10 genes/loci that had a summary P value < 0.05. Sensitivity analysis indicated that the results were robust. Replication studies and understanding the roles of these genes in keratoconus are warranted.

A variety of factors have been linked to the occurrence of anterior cruciate ligament injury (ACLI), including sex, familial factors, and genetic variations. To find the genetic loci associated with ACLI and explore the genetic mechanism of ACLI in order to provide a genetic basis for the diagnosis, prognosis, and treatment of patients with ACLI. Cross-sectional study. Hospital. Data from 101 Chinese Yunnan Han patients with ACLI and 110 Yunnan Han individuals without ACLI (control group) were collected. The single nucleotide polymorphisms of rs1800012, rs12722 and rs13946, rs970547 and rs240736 and the rs1800787, rs1800788, rs1800789, rs1800790, rs1800791, and rs2227389 in the ( ) promoter region were analyzed using restriction fragment length polymorphism and DNA sequencing detection, and their genetic associations with ACLI were assessed. Single nucleotide polymorphisms of rs1800012, rs12722 and rs13946, and the rs1800789 and rs1800791 in the promoter region showed no difference between patients with ACLI and control participants, but the changes of rs970547 and rs240736 and the rs1800787, rs1800788, rs1800790, and rs2227389 genotypes in the promoter region were associated with ACLI. Furthermore, the rs970547 allele and genotype frequencies in male ACLI patients were different from the control group ( < .05): the frequencies of the rs970547 A and G alleles in the patients were 71.9% and 28.1%, respectively, and in the control group were 58.8% and 41.2%, respectively. The frequencies of AA, AG, and GG genotypes in the patients were 49.3%, 45.2%, and 5.5%, respectively, and in the control group were 27.5%, 62.7%, and 9.8%, respectively, suggesting that male carriers of rs970547 A and rs970547 AA were at high risk of ACLI. Males with the rs970547 A allele and rs970547 AA genotype of may be at high risk for ACLI. Low rs1800787 TT and high rs1800788 CT, rs1800790 AG, and rs2227389 CT frequencies as well as high TGA of rs1800790, rs1800791, and rs2227389 in the promoter region may be genetic risk factors related to ACLI.