Extracellular matrix proteins play a crucial role in influencing the invasion of trophoblast cells. However the role of collagens and collagen type IV (col-IV) in particular at the implantation site is not clear.

Alport syndrome is a hereditary type IV collagen disease leading to progressive renal fibrosis, hearing loss and ocular changes. End stage renal failure usually develops during adolescence. COL4A3-/- mice serve as an animal model for progressive renal scarring in Alport syndrome. The present study evaluates the role of Discoidin Domain Receptor 1 (DDR1) in cell-matrix interaction involved in pathogenesis of Alport syndrome including renal inflammation and fibrosis. DDR1/COL4A3 Double-knockouts were compared to COL4A3-/- mice with 50% or 100% expression of DDR1, wildtype controls and to DDR1-/- COL4A3+/+ controls for over 6years. Double-knockouts lived 47% longer, mice with 50% DDR1 lived 29% longer and showed improved renal function (reduction in proteinuria and blood urea nitrogen) compared to animals with 100% DDR1 expression. Loss of DDR1 reduced proinflammatory, profibrotic cells via signaling of TGFbeta, CTGF, NFkappaB and IL-6 and decreased deposition of extracellular matrix. Immunogold-staining and in-situ hybridisation identified podocytes as major players in DDR1-mediated fibrosis and inflammation within the kidney. In summary, glomerular epithelial cells (podocytes) express DDR1. Loss of DDR1-expression in the kidney delayed renal fibrosis and inflammation in hereditary type IV collagen disease. This supports our hypothesis that podocyte-matrix interaction via collagen receptors plays an important part in progression of renal fibrosis in Alport disease. The blockade of collagen-receptor DDR1 might serve as an important new therapeutic concept in progressive fibrotic and inflammatory diseases in the future.

Type IV collagen with a triple-helical structure composed of three α chains is a major component of basement membrane. Previously, we reported that non-triple helical form of type IV collagen α1 chain (NTHα1(IV)) was isolated from human placenta and the culture media of human cells. In the present study, we report on the localization of NTH α1(IV) with a monoclonal antibody #370, exclusively reactive for the nascent chain, in the rabbit tissues. The staining was found on the basement membrane of blood vessels, of endomysium, of nerve, and of kidney but not on epithelial basement membrane. In a rabbit angiogenic model, #370 antibody staining was exclusively observed in neovascular tip region of endothelial cells, where no staining with anti-type IV collagen antibody was seen. Distinct localizations suggest that NTHα1(IV) is produced and stably deposited in endothelial cells and the surroundings under physiological conditions with some physiological roles in relation to the dynamics of vascular system.

Basement membrane (BM) matrices surround and separate most tissues. However, through poorly understood mechanisms, BMs of adjacent tissue can also stably link to support organ structure and function. Using endogenous knock-in fluorescent proteins, conditional RNAi, optogenetics, and quantitative live imaging, we identified extracellular matrix proteins mediating a BM linkage (B-LINK) between the uterine utse and epidermal seam cell BMs in Caenorhabditis elegans that supports the uterus during egg-laying. We found that hemicentin is secreted by the utse and promotes fibulin-1 assembly to jointly initiate the B-LINK. During egg-laying, however, both proteins' levels decline and are not required for B-LINK maintenance. Instead, we discovered that hemicentin recruits ADAMTS9/20, which facilitates the assembly of high levels of type IV collagen that sustains the B-LINK during the mechanically active egg-laying period. This work reveals mechanisms underlying BM-BM linkage maturation and identifies a crucial function for hemicentin and fibulin-1 in initiating attachment and type IV collagen in strengthening this specialized form of tissue linkage.

No reliable, non-invasive biomarker of metastatic breast cancer (mBC) exists: circulating CA15-3 (cCA15-3) is the marker mostly used to monitor mBC. Circulating collagen IV (cCOLIV) has been evaluated in other metastatic cancers and has been found to be a promising biomarker. The overarching aim of this study was to evaluate cCOLIV as a potential biomarker in patients with mBC. The first aim was to determine the levels of cCOL IV and cCA15-3 in patients with healthy controls, primary breast cancer (pBC) and mBC. The second aim was to compare levels of cCOLIV and cCA15-3 in patients with different metastatic sites of BC. The third aim was to investigate the prognostic value of cCOLIV and cCA15-3 for mBC patients. The fourth aim was to analyse whether a combination of the two biomarkers was more accurate in detecting mBC than a single marker. Lastly, we investigated the tissue expression levels of COLIV in BC bone metastases (BM) and liver metastases (LM). Plasma levels of cCOLIV and cCA15-3 from healthy controls and patients with pBC and mBC were measured. COLIV expression in tissue from patients with LM and BM was analysed using immunohistochemistry. Clinical and survival data were collected from medical charts. The levels of cCOLIV and cCA15-3 were significantly elevated in mBC patients compared with healthy controls and pBC patients. No differences in cCOLIV and cCA15-3 levels were found based on the metastatic site. High levels of cCOLIV, but not cCA15-3, correlated with poorer survival. cCOLIV alone and the combination of cCA15-3 and cCOLIV were superior to cCA15-3 at detecting mBC. COL IV was highly expressed in the tissue of LM and BM. Our study suggests that cCOLIV is a potential marker to monitor patients with BC.

In X-linked Alport syndrome, mutations in the COL4A5 gene encoding the alpha 5 chain of type IV collagen result in progressive renal failure. This nephropathy appears to relate to the arrest of a switch from an alpha 1/alpha 2 to an alpha 3/alpha 4/alpha 5 network of type IV collagen in the developing glomerular basement membrane (GBM; Kalluri et al, J Clin Invest 99:2470, 1997).

Alport syndrome (AS) is a hereditary glomerulonephritis caused by COL4A3, COL4A4 or COL4A5 gene mutations and characterized by abnormalities of glomerular basement membranes (GBMs). Due to a lack of curative treatments, the condition proceeds to end-stage renal disease even in adolescents. Hampering drug discovery is the absence of effective in vitro methods for testing the restoration of normal GBMs. Here, we aimed to develop kidney organoid models from AS patient iPSCs for this purpose. We established iPSC-derived collagen α5(IV)-expressing kidney organoids and confirmed that kidney organoids from COL4A5 mutation-corrected iPSCs restore collagen α5(IV) protein expression. Importantly, our model recapitulates the differences in collagen composition between iPSC-derived kidney organoids from mild and severe AS cases. Furthermore, we demonstrate that a chemical chaperone, 4-phenyl butyric acid, has the potential to correct GBM abnormalities in kidney organoids showing mild AS phenotypes. This iPSC-derived kidney organoid model will contribute to drug discovery for AS.

Pancreas cancer is a dreaded disease with high mortality, despite progress in surgical and oncological treatments in recent years. The field is hampered by a lack of good prognostic and predictive tumour biomarkers to be used during follow-up of patients.

The plasma concentration of 5-hydroxytryptamine (5-HT) in diabetic patients is higher than that in normal subjects. Since recent reports have demonstrated the presence of 5-HT2A receptor in glomerular mesangial cells, it is possible that 5-HT may be involved in the development of diabetic nephropathy through the 5-HT2A receptor in mesangial cells. Because expansion of the glomerular mesangial lesion is a characteristic feature of diabetic nephropathy, we examined the effect of 5-HT on the production of type IV collagen by human mesangial cells.

Mucin 5AC (MUC5AC) is excessively secreted in the respiratory tract of patients with asthma. Suppressing this secretion is important for improving the air passages, which facilitates easy breathing. We have previously reported that the addition of type IV collagen, a typical extracellular matrix (ECM) protein, to the culture medium for human cell lines and primary cells reduced MUC5AC secretion. In this report, we further investigated the effect of type IV collagen on MUC5AC secretion in vivo. We employed ovalbumin (OVA)-sensitized mice to model of asthma and exposed them to type IV collagen to verify the reducing effect of MUC5AC in vivo. The amount of MUC5AC in bronchoalveolar lavage fluid was examined after nebulization of type IV collagen. Hypersecretion of MUC5AC of the OVA-sensitized mice was suppressed by type IV collagen exposure in a time- and dose-dependent manner. Furthermore, type IV collagen exposure to OVA-sensitized mice decreased integrin α2 and β1 expression in the lungs and increased the levels of Akt and extracellular signal-regulated kinase (ERK) phosphorylation in the trachea. These results suggest that type IV collagen suppresses MUC5AC hypersecretion via modulating integrin expression and Akt/ERK phosphorylation in the respiratory tract of the OVA-sensitized mice.

Basement membranes (BMs) are cell-associated extracellular matrices that support tissue integrity, signaling, and barrier properties. Type IV collagen is critical for BM function, yet how it is directed into BMs in vivo is unclear. Through live-cell imaging of endogenous localization, conditional knockdown, and misexpression experiments, we uncovered distinct mechanisms of integrin-mediated collagen recruitment to Caenorhabditis elegans postembryonic gonadal and pharyngeal BMs. The putative laminin-binding αINA-1/βPAT-3 integrin was selectively activated in the gonad and recruited laminin, which directed moderate collagen incorporation. In contrast, the putative Arg-Gly-Asp (RGD)-binding αPAT-2/βPAT-3 integrin was activated in the pharynx and recruited high levels of collagen in an apparently laminin-independent manner. Through an RNAi screen, we further identified the small GTPase RAP-3 (Rap1) as a pharyngeal-specific PAT-2/PAT-3 activator that modulates collagen levels. Together, these studies demonstrate that tissues can use distinct mechanisms to direct collagen incorporation into BMs to precisely control collagen levels and construct diverse BMs.

The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BMfunctions due to aberrant COL4A1 protein, we have carried out a series of tests using Drosophila DTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, Diptericin B, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that aberrant structure and function of the COL4A1 protein may also be a significant factor in modulating immunity.

The mechanisms that generate specific neuronal connections in the brain are under intense investigation. In zebrafish, retinal ganglion cells project their axons into at least six layers within the neuropil of the midbrain tectum. Each axon elaborates a single, planar arbor in one of the target layers and forms synapses onto the dendrites of tectal neurons. We show that the laminar specificity of retinotectal connections does not depend on self-sorting interactions among RGC axons. Rather, tectum-derived Slit1, signaling through axonal Robo2, guides neurites to their target layer. Genetic and biochemical studies indicate that Slit binds to Dragnet (Col4a5), a type IV Collagen, which forms the basement membrane on the surface of the tectum. We further show that radial glial endfeet are required for the basement-membrane anchoring of Slit. We propose that Slit1 signaling, perhaps in the form of a superficial-to-deep gradient, presents laminar positional cues to ingrowing retinal axons.

An early feature of diabetic nephropathy is the alteration of the glomerular basement membrane (GBM), which may result in microalbuminuria, subsequent macroproteinuria, and eventual chronic renal failure. Although type IV collagen is the main component of thickened GBM in diabetic nephropathy, cellular metabolism of each alpha chains of type IV collagen has not been well studied. To investigate the regulation of alpha(IV) chains in diabetic conditions, we examined whether glucose and advanced glycosylation endproduct (AGE) regulate the metabolism of each alpha(IV) chains in the diabetic tissue and glomerular epithelial cells (GEpC). Glomerular collagen alpha3(IV) and alpha5(IV) chains protein were higher and more intense in immunofluorescence staining according to diabetic durations compared to controls. In vitro, mainly high glucose and partly AGE usually increased total collagen protein of GEpC by [(3)H]-proline incorporation assay and each alpha(IV) chain proteins including alpha1(IV), alpha3(IV), and alpha5(IV) in time-dependent and subchain-specific manners. However, the changes of each alpha(IV) chains mRNA expression was not well correlated to the those of each chain proteins. The present findings suggest that the metabolism of individual alpha(IV) chains of GBM is differentially regulated in diabetic conditions and those changes might be induced not only by transcriptional level but also by post-translational modifications.

Type IV collagen is the main component of the basement membrane that gives strength to the blood-gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown.

Circulating type IV collagen (cCOL IV) is a potential biomarker for patients with colorectal liver metastases (CLM) who present with elevated levels of COL IV in both CLM tissue and circulation. This study aimed to establish the cellular origin of elevated levels of COL IV and analyze circulating COL IV in CLM patients. The cellular source was established through in situ hybridization, immunohistochemical staining, and morphological evaluation. Cellular expression in vitro was assessed by immunofluorescence. Tissue expression of COL IV-degrading matrix metalloproteinases (MMPs)-2, -7, -9, and -13 was studied with immunohistochemical staining. Plasma levels of COL IV in CLM patients and healthy controls were analyzed with ELISA. This study shows that cancer-associated fibroblasts (CAFs) express COL IV in the stroma of CLM and that COL IV is expressed in vitro by fibroblasts but not by tumor cells. MMP-2, -7, -9, and -13 are expressed in CLM tissue, mainly by hepatocytes and immune cells, and circulating COL IV is significantly elevated in CLM patients compared with healthy controls. Our study shows that stromal cells, not tumor cells, produce COL IV in CLM, and that circulating COL IV is elevated in patients with CLM.

During adipogenic differentiation human mesenchymal stem cells (hMSC) produce collagen type IV. In immunofluorescence staining differentiating hMSCs started to express collagen type IV when Oil Red O-positive fat droplets appeared intracellularly. Quantitative real time-polymerase chain reaction confirmed progressive increase of collagen type IV α1 and α2 mRNA levels over time, 18.6- and 12.2-fold by day 28, respectively, whereas the copy numbers of α3-α6 mRNAs remained rather stable and low. Type IV collagen was in confocal laser scanning microscopy seen around adipocytes, where also laminins and nidogen were found, suggesting pericellular deposition of all key components of the fully developed basement membrane. Immunofluorescence staining of matrix metalloproteinase-2 (MMP-2, 72 kD type IV collagenase, gelatinase A) and MMP-9 (92 kD type IV collagenase, gelatinase B) disclosed only faint staining of MSCs, but MMP-9 was strongly induced during adipogenesis, whereas MSC supernatants disclosed in zymography pro-MMP-2 and faint pro-MMP-9 bands, which increased over time, with partial conversion of pro-MMP-2 to its active 62 kD form. Differentiation was associated with increasing membrane type 1-MMP/MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) staining, which may enable participation of type IV collagenases in basement membrane remodelling via ternary MT1-MMP/TIMP-2/MMP-2 or -9 complexes, focalizing the fully active enzyme to the cell surface. MMP-9, which increased more in immunofluorescence staining, was perhaps preferentially bound to cell surface and/or remodelling adipocyte basement membrane. These results suggest that upon MSC-adipocyte differentiation collagen type IV synthesis and remodelling become necessary when intracellular accumulation of fat necessitates a dynamically supporting and instructive, partly denatured adipogenic pericellular type IV collagen scaffold.

Progressive glomerulonephritis (GN) is characterized by an excessive accumulation of extracellular (ECM) proteins, mainly type IV collagen (COLIV), in the glomerulus leading to glomerulosclerosis. The current therapeutic approach to GN is suboptimal. Epigenetic drugs could be novel therapeutic options for human disease. Among these drugs, bromodomain and extra-terminal domain (BET) inhibitors (iBETs) have shown beneficial effects in experimental kidney disease and fibrotic disorders. Sex-determining region Y-box 9 (SOX9) is a transcription factor involved in regulating proliferation, migration, and regeneration, but its role in kidney fibrosis is still unclear. We investigated whether iBETs could regulate ECM accumulation in experimental GN and evaluated the role of SOX9 in this process. For this purpose, we tested the iBET JQ1 in mice with anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS). In NTS-injected mice, JQ1 treatment reduced glomerular ECM deposition, mainly by inhibiting glomerular COLIV accumulation and Col4a3 gene overexpression. Moreover, chromatin immunoprecipitation assays demonstrated that JQ1 inhibited the recruitment and binding of BRD4 to the Col4a3 promoter and reduced its transcription. Active SOX9 was found in the nuclei of glomerular cells of NTS-injured kidneys, mainly in COLIV-stained regions. JQ1 treatment blocked SOX9 nuclear translocation in injured kidneys. Moreover, in vitro JQ1 blocked TGF-β1-induced SOX9 activation and ECM production in cultured mesangial cells. Additionally, SOX9 gene silencing inhibited ECM production, including COLIV production. Our results demonstrated that JQ1 inhibited SOX9/COLIV, to reduce experimental glomerulosclerosis, supporting further research of iBET as a potential therapeutic option in progressive glomerulosclerosis.

Type IV collagens (Col IV), components of basement membrane, are essential in the maintenance of tissue integrity and proper function. Alteration of Col IV is related to developmental defects and diseases, including cancer. Col IV α chains form α1α1α2, α3α4α5 and α5α5α6 protomers that further form collagen networks. Despite knowledge on the functions of major Col IV (α1α1α2), little is known whether minor Col IV (α3α4α5 and α5α5α6) plays a role in cancer. It also remains to be elucidated whether major and minor Col IV are functionally redundant. We show that minor Col IV α5 chain is indispensable in cancer development by using α5(IV)-deficient mouse model. Ablation of α5(IV) significantly impeded the development of KrasG12D-driven lung cancer without affecting major Col IV expression. Epithelial α5(IV) supports cancer cell proliferation, while endothelial α5(IV) is essential for efficient tumor angiogenesis. α5(IV), but not α1(IV), ablation impaired expression of non-integrin collagen receptor discoidin domain receptor-1 (DDR1) and downstream ERK activation in lung cancer cells and endothelial cells. Knockdown of DDR1 in lung cancer cells and endothelial cells phenocopied the cells deficient of α5(IV). Constitutively active DDR1 or MEK1 rescued the defects of α5(IV)-ablated cells. Thus, minor Col IV α5(IV) chain supports lung cancer progression via DDR1-mediated cancer cell autonomous and non-autonomous mechanisms. Minor Col IV can not be functionally compensated by abundant major Col IV.

Pathogenic variants in type IV collagen have been reported to account for a significant proportion of chronic kidney disease. Accordingly, genetic testing is increasingly used to diagnose kidney diseases, but testing also may reveal rare missense variants that are of uncertain clinical significance. To aid in interpretation, computational prediction (called in silico) programs may be used to predict whether a variant is clinically important. We evaluate the performance of in silico programs for COL4A3/A4/A5 variants.