Raghuram Kalluri

Early role of Fsp1 in epithelial-mesenchymal transformation

A seamless plasticity exists among cells shifting between epithelial and mesenchymal phenotypes during early development and again later, in adult tissues, following wound repair or organ remodeling in response to injury. Fsp1, a gene encoding a fibroblast-specific protein associated with mesenchymal cell morphology and motility, is expressed during epithelial-mesenchymal transformations (EMT) in vivo. In the current study, we identified several cytokines that induce Fsp1 in cultured epithelial cells. A combination of these factors, however, was most efficacious at completing the process of EMT. The optimal combination identified were two of the cytokines classically associated with fibrosis, i.e., transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). To confirm that it was the induction of Fsp1 by these cytokines mediating EMT, we used antisense oligomers to block Fsp1 production and subsequently measured cell motility and markers of EMT phenotype. The antisense oligomers suppressed Fsp1 expression and epithelial transformation; therefore, we conclude that the appearance of Fsp1 is an important early event in the pathway toward EMT.

Isoform switching of type IV collagen is developmentally arrested in X-linked Alport syndrome leading to increased susceptibility of renal basement membranes to endoproteolysis.

Normal glomerular capillaries filter plasma through a basement membrane (GBM) rich in alpha3(IV), alpha4(IV), and alpha5(IV) chains of type IV collagen. We now show that these latter isoforms are absent biochemically from the glomeruli in patients with X-linked Alport syndrome (XAS). Their GBM instead retain a fetal distribution of alpha1(IV) and alpha2(IV) isoforms because they fail to developmentally switch their alpha-chain use. The anomalous persistence of these fetal isoforms of type IV collagen in the GBM in XAS also confers an unexpected increase in susceptibility to proteolytic attack by collagenases and cathepsins. The incorporation of cysteine-rich alpha3(IV), alpha4(IV), and alpha5(IV) chains into specialized basement membranes like the GBM may have normally evolved to protectively enhance their resistance to proteolytic degradation at the site of glomerular filtration. The relative absence of these potentially protective collagen IV isoforms in GBM from XAS may explain the progressive basement membrane splitting and increased damage as these kidneys deteriorate.

Susceptibility to anti-glomerular basement membrane disease and Goodpasture syndrome is linked to MHC class II genes and the emergence of T cell-mediated immunity in mice.

We developed a new mouse model of human anti-glomerular basement membrane (GBM) disease to better characterize the genetic determinants of cell-mediated injury. While all major histocompatibility complex (MHC) haplotypes (H-2a, k, s, b, and d) immunized with alpha3 NC1 domains of type IV collagen produce anti-alpha3(IV) NC1 antibodies that cross-react with human Goodpasture [anti-GBM/anti-alpha3(IV) NC1] autoantibodies, only a few strains developed nephritis and lung hemorrhage associated with Goodpasture syndrome. Crescentic glomerulonephritis and lung hemorrhage were MHC-restricted in haplotypes H-2s, b, and d (A beta/A alpha region in H-2s) and associated with the emergence of an IL-12/Th1-like T cell phenotype. Lymphocytes or anti-alpha3(IV) NC1 antibodies from nephritogenic strains transfer disease to syngeneic recipients. However, passive transfer of isogenic alpha3(IV) NC1 antibodies into -/- T cell receptor-deficient mice failed to produce nephritis. Finally, nephritis and its associated IL-12/Th1-like T cell response attenuate in disease-susceptible mice tolerized orally to alpha3(IV) collagen before immunization. Our findings suggest collectively, as a hypothesis, that anti-GBM antibodies in mice only facilitate disease in MHC haplotypes capable of generating nephritogenic lymphocytes with special T cell repertoires.

Seminiferous Tubule Basement Membrane COMPOSITION AND ORGANIZATION OF TYPE IV COLLAGEN CHAINS, AND THE LINKAGE OF α3(IV) AND α5(IV) CHAINS

Seminiferous tubule basement membrane (STBM) plays an important role in spermatogenesis. In the present study, the composition and structural organization of type IV collagen of bovine STBM was investigated. STBM was found to be composed of all six α-chains of type IV collagen based upon immunocytochemical and biochemical analysis. The content of α3(IV) chain (40%) and the α4(IV) chain (18%) was substantially higher than in any other basement membrane collagen. The supramolecular structure of the six α(IV) chains was investigated using pseudolysin (EC 3.4.24.26) digestion to excise triple-helical molecules, subsequent collagenase digestion to produce NC1 hexamers and antibody affinity chromatography to resolve populations of NC1 hexamers. The hexamers, which reflect specific arrangements of α(IV) chains, were characterized for their α(IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with α(IV) chain-specific antibodies. Three major hexamer populations were found that represent the classical network of the α1(IV) and α2(IV) chains and two novel networks, one composed of the α1(IV)-α6(IV) chains and the other composed of the α3(IV)-α6(IV) chains. The results establish a structural linkage between the α3(IV) and α5(IV) chains, suggesting a molecular basis for the conundrum in which mutations in the gene encoding the α5(IV) chain cause defective assembly of the α3(IV) chain in the glomerular basement membrane of patients with Alport syndrome.

Coordinate Gene Expression of the α3, α4, and α5 Chains of Collagen Type IV EVIDENCE FROM A CANINE MODEL OF X-LINKED NEPHRITIS WITH A COL4A5 GENE MUTATION

Canine X-linked hereditary nephritis is an animal model for human X-linked hereditary nephritis with a premature stop codon in the α5(IV) gene of collagen type IV. We used this model to examine the other α(IV) chains at the mRNA and protein level in the kidney, since in human X-linked hereditary nephritis, the α3(IV) and α4(IV) chains are often absent from the glomerular basement membrane, although both are encoded by autosomal genes. cDNA probes for the α1(IV)-α6(IV) chains were generated from normal dog kidney using the polymerase chain reaction. Sequences were ≥88% identical at the DNA level and ≥92% identical at the protein level to the respective human α(IV) chains. By Northern analysis, transcripts for the α1(IV), α2(IV), and α6(IV) chains were detected at comparable levels in both normal and affected male dog kidney RNA. As previously shown, the transcript for the α5(IV) chain was reduced to ~10% of normal. Unexpectedly, the α3(IV) and α4(IV) transcripts were both decreased ≥77% in affected male dog kidney, suggesting a mechanism coordinating the expression of these three basement membrane components. The NC1 domain of collagen type IV isolated from normal dog glomeruli was positive for the α3(IV), α4(IV), and α5(IV) chains by Western blotting. In contrast, in the NC1 domain isolated from affected dog glomeruli, these three chains were not detectable, except for a trace of α3(IV) dimer. In X-linked hereditary nephritis, the absence of the α3(IV) and α4(IV) chains from glomerular basement membrane may reflect factors acting at the transcriptional and/or translational level in addition to the protein assembly level.

Identification and Localization of Type IV Collagen Chains in the Inner Ear Cochlea

Mutations in the genes encoding the alpha3(IV), alpha4(IV) and alpha5(IV) chains of type IV collagen have been implicated in the pathogenesis of Alports syndrome, a hereditary disorder characterized by progressive nephropathy and sensorineural deafness. The known expression of these chains in kidney basement membranes supports the contention that they play a crucial role in the ultrafiltration function. Whether they play a role in auditory signal transduction remains unknown as heretofore, they have not been identified in the inner ear. In the present study, the expression of type IV collagen in cochlea of the inner ear of guinea pigs was determined. All six alpha-chains of type IV collagen were identified by biochemical and immunological methods. By indirect immunofluorescence, alpha1(IV) and alpha2(IV) chains were localized to the spiral limbus, basilar membrane and tectorial membrane. The alpha3(IV), alpha4(IV), alpha5(IV) and alpha6(IV) chains localized exclusively to the tectorial membrane and basilar membrane. These results suggest a possible role of type IV collagen chains in the active tuning of the basilar and tectorial membrane, an essential step in frequency discrimination and amplification of auditory signals.

Structure and composition of type IV collagen of bovine aorta

To determine the chain composition of type IV collagen of bovine thoracic aorta, we analyzed collagenase-solubilized carboxyl-terminal noncollagenous (NC1)-domains by high-pressure liquid chromatography, two-dimensional electrophoresis, immunoblotting and enzyme-linked immunoassay. In addition to the classical alpha 1- and alpha 2-chains, we found small amounts of the recently discovered alpha 3-, alpha 4- and alpha 5-chains. The alpha 3- and alpha 4-chains were, collectively, 7-13% of the total, and the alpha 5-chain was present in a low amount. Seventy-nine percent of the NC1-domains were dimerized. Immunolocalization studies on sections of aorta showed that the alpha 3- and alpha 5-chains were present, along with alpha 1- and alpha 2-chains, in the subendothelium and media. In capillaries of the media, the alpha 3-chain was found at relatively high levels and was co-localized with alpha 1- and alpha 2-chains. Digestion of aorta with Pseudomonas aeruginosa elastase yielded soluble multimolecular assemblies of type IV collagen. Electron microscopy results provided a direct demonstration of the supramolecular structure, in which the collagen molecules were tetramerized at the amino-terminal end and dimerized at the carboxyl-terminal end.

Pathology of glomerular basement membrane nephropathy.

Studies of the molecular pathology of glomerular basement membrane have led to the discovery of four novel chains of type IV collagen, the latest of which is the alpha 6(IV) chain. Three chains have been shown to be directly involved in the pathogenesis of Goodpasture syndrome, Alport syndrome, and Alport posttransplantation antiglomerular basement membrane nephritis. This review focuses on the novel chains with respect to recent advances in their structure, gene organization, and involvement in the pathogenesis of these glomerulopathies.