Deletion of CD151 results in a strain-dependent glomerular disease due to severe alterations of the glomerular basement membrane.

Alterations in CD151 have been associated with primary glomerular disease in both humans and mice, implicating CD151 as a key component of the glomerular filtration barrier. CD151 belongs to the tetraspanin family and associates with cell-matrix adhesion complexes such as alpha3beta1-integrin. Here we show that Cd151-deficient mice develop severe kidney disease on an FVB background but are healthy on a B6 background, providing a new and unique tool for the identification of genes that modulate the onset of proteinuria. To better understand the function of CD151 in the kidney, we studied its expression pattern and characterized early ultrastructural defects in Cd151-null kidneys. CD151 is expressed in podocytes of the mouse kidney and co-localizes with alpha3-integrin at the base of podocyte foot processes, at the site of anchorage to the glomerular basement membrane (GBM). Interestingly, the first ultrastructural lesions seen at the onset of proteinuria in Cd151-null kidneys were severe alterations of the GBM, reminiscent of Alport syndrome and consisting of massive thickening and splitting of the GBM. These lesions are associated with increased expression of GBM components. Podocyte abnormalities, effacement of foot processes, and podocyte loss appear to occur consequently to the GBM damage. In conclusion, CD151 appears to be involved in the establishment, maturation, and/or maintenance of the GBM structure in addition to its role in integrin-mediated adhesion strengthening.

The role of the tetraspanin CD151 in primary keratinocyte and fibroblast functions: implications for wound healing.

Previous studies showed that CD151-null mice have a skin wound healing deficit. To gain an understanding of the role of CD151 in re-epithelialisation and dermal contraction, keratinocyte and fibroblast functions were assayed. Primary CD151-null keratinocytes displayed defective migration on Matrigel (a basement membrane equivalent) and laminin-332, the primary adhesion component of basement membranes, but not on collagen-I. Adhesion, spreading and proliferation were also deficient on laminin-332, but not collagen-I. The data suggest that loss of CD151 impairs the function of its primary interaction partners, integrin alpha3beta1- and/or alpha6beta4 which bind to laminin-332. Skin fibroblasts also produce CD151 mRNA. CD151-null fibroblasts migrated significantly faster on collagen I than wild type fibroblasts, confirming that they possess functional collagen receptors. However, no significant decrease in the ability of CD151-null fibroblasts to cause contraction in floating collagen gel assays in response to transforming growth factor beta-1 (TGF-beta1) or platelet derived growth factor (PDGF-BB) was observed, nor was there an effect on fibroblast adhesion or proliferation on collagen-I. The data implicate CD151 as a facilitator of laminin-332-mediated keratinocyte functions that impact on the re-epithelialisation process intrinsic to wound healing and further suggest a potential novel role for CD151 in fibroblast migration.

Resistance to c-KIT kinase inhibitors conferred by V654A mutation.

Certain mutations within c-KIT cause constitutive activation of the receptor and have been associated with several human malignancies. These include gastrointestinal stromal tumors (GIST), mastocytosis, acute myelogenous leukemia, and germ cell tumors. The kinase inhibitor imatinib potently inhibits c-KIT and is approved for treatment of GIST. However, secondary point mutations can develop within the kinase domain to confer resistance to imatinib and cause drug-resistant relapse. A common mutation, which results in a V654A substitution, has been documented in imatinib-resistant GIST patients. We expressed c-KIT cDNA constructs encoding the V654A substitution alone and in combination with a typical activating exon 11 mutation characteristic of GIST, V560G, in factor-dependent FDC-P1 cells. The V654A substitution alone resulted in enhanced proliferation in c-KIT ligand (stem cell factor) but not factor independence. Cells expressing the double mutant were, like those expressing single V560G mutant c-KIT, factor independent. Analysis of cellular proliferation in the presence of imatinib showed that the V654A substitution alone conferred resistance. The difference in sensitivity was especially pronounced for cells expressing single mutant V560G c-KIT compared with double mutant V560G/V654A c-KIT. The findings were supported by studies of c-KIT phosphorylation. Analysis of the crystal structure of imatinib in complex with the kinase domain of c-KIT predicts that the V654A substitution directly affects the binding of imatinib to the receptor. Alternative c-KIT inhibitors, nilotinib (AMN107) and PKC412, were also less active on V560G/V654A c-KIT than on the V560G single mutant; however, nilotinib, like imatinib, potently inhibited the V560G mutant. PKC412 strongly inhibited imatinib-resistant D816V c-KIT.

Vitamin A regulation of BMP4 expression in the male germ line.

The molecular mechanisms leading to male infertility in vitamin A deficient (VAD) rodents have never been fully elucidated. Here, we report an interaction between BMP4 and retinoid signaling pathways in germ cells that may help clarify the biochemical basis of VAD. Adult germ cells, in particular spermatogonia, expressed BMP4 at both the mRNA and protein levels. BMP4 expression was significantly up-regulated in the testes of VAD mice and was down-regulated in freshly isolated germ cells and VAD testes by retinol, but not retinoic acid. The retinoid-responsive gene, RARbeta, was not induced in germ cells following retinoid treatment. Examination of BMP4 promoter usage in spermatogonia and the VAD testis revealed that germ cells utilize the recently characterized BMP4 intron 2 promoter, in addition to the classical 1A and 1B promoters. The observed decrease in BMP4 in response to retinol was mediated by the 1A and intron 2 promoters of the BMP4 gene. Our results reflect a direct requirement for retinoids by germ cells for the resumption of spermatogenesis in VAD animals via mechanisms that involve the suppression of BMP4 expression.

Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition.

Mammalian spermatozoa undergo a series of molecular and biochemical changes collectively termed capacitation prior to acquiring the ability to fertilise the oocyte. Although phosphorylation of sperm proteins on tyrosine residues has been recognised as an important component of this process, the precise relationship between the phosphorylation status of mammalian spermatozoa and their capacity for fertilisation has remained unclear. In this study we demonstrate a causal relationship between tyrosine phosphorylation in spermatozoa and sperm-zona interaction. The phosphotyrosine expression associated with sperm capacitation localised to internal flagellar structures in permeabilised cells but could also be detected on the exterior surface of the sperm head in live cells. Importantly, almost all spermatozoa bound to the zona pellucida demonstrated this pattern of phosphoprotein localisation, compared to fewer than 15% of the free-swimming population. These data suggest that tyrosine phosphorylation plays a significant role in remodelling the sperm surface, so that these cells are able to recognise the zona pellucida. Phosphoproteome analysis yielded the first evidence of molecular chaperones, endoplasmin (erp99) and heat shock protein 60 (hsp60), as targets for phosphorylation on the surface of mouse spermatozoa, whereas immunofluorescence localised these proteins to the precise region of the sperm head that participates in zona recognition. Based on these results, we propose a novel mechanism for mammalian gamete interaction whereby the activation of sperm-surface chaperones by tyrosine phosphorylation during capacitation may trigger conformational changes facilitating the formation of a functional zona pellucida receptor complex on the surface of mammalian spermatozoa.