Absence of Dap12 and the αvß3 integrin causes severe osteopetrosis.

In vitro, ligand occupancy of αvß3 integrin induces phosphorylation of Dap12, which is essential for osteoclast function. Like mice deleted of only αvß3, Dap12(-/-) mice exhibited a slight increase in bone mass, but Dap12(-/-) mice, lacking another ITAM protein, FcRγ, were severely osteopetrotic. The mechanism by which FcRγ compensates for Dap12 deficiency is unknown. We find that co-deletion of FcRγ did not exacerbate the skeletal phenotype of ß3(-/-) mice. In contrast, ß3/Dap12 double-deficient (DAP/ß3(-/-)) mice (but not ß1/Dap12 double-deficient mice) were profoundly osteopetrotic, reflecting severe osteoclast dysfunction relative to those lacking αvß3 or Dap12 alone. Activation of OSCAR, the FcRγ co-receptor, rescued Dap12(-/-) but not DAP/ß3(-/-)osteoclasts. Thus, the absence of αvß3 precluded compensation for Dap12 deficiency by FcRγ. In keeping with this, Syk phosphorylation did not occur in OSCAR-activated DAP/ß3(-/-) osteoclasts. Thus, FcRγ requires the osteoclast αvß3 integrin to normalize the Dap12-deficient skeleton.

Integrin αvß3 drives slug activation and stemness in the pregnant and neoplastic mammary gland.

Although integrin αvß3 is linked to cancer progression, its role in epithelial development is unclear. Here, we show that αvß3 plays a critical role in adult mammary stem cells (MaSCs) during pregnancy. Whereas αvß3 is a luminal progenitor marker in the virgin gland, we noted increased αvß3 expression in MaSCs at midpregnancy. Accordingly, mice lacking αvß3 or expressing a signaling-deficient receptor showed defective mammary gland morphogenesis during pregnancy. This was associated with decreased MaSC expansion, clonogenicity, and expression of Slug, a master regulator of MaSCs. Surprisingly, αvß3-deficient mice displayed normal development of the virgin gland with no effect on luminal progenitors. Transforming growth factor ß2 (TGF-ß2) induced αvß3 expression, enhancing Slug nuclear accumulation and MaSC clonogenicity. In human breast cancer cells, αvß3 was necessary and sufficient for Slug activation, tumorsphere formation, and tumor initiation. Thus, pregnancy-associated MaSCs require a TGF-ß2/αvß3/Slug pathway, which may contribute to breast cancer progression and stemness.

Endogenous ribosomal protein L29 (RPL29): a newly identified regulator of angiogenesis in mice.

Cellular ribosomal protein L29 (RPL29) is known to be important in protein synthesis, but its function during angiogenesis has never been described before. We have shown previously that mice lacking ß3-integrins support enhanced tumour angiogenesis and, therefore, deletion of endothelial αvß3 can provide a method for discovery of novel regulators of tumour angiogenesis. Here, we describe significant upregulation of RPL29 in ß3-null endothelial cells at both the mRNA and protein level. Ex vivo, we show that VEGF-stimulated microvessel sprouting was reduced significantly in Rpl29-heterozygous and Rpl29-null aortic ring assays compared with wild-type controls. Moreover, we provide in vivo evidence that RPL29 can regulate tumour angiogenesis. Tumour blood vessel density in subcutaneously grown Lewis lung carcinomas was reduced significantly in Rpl29-mutant mice. Additionally, depletion of Rpl29 using RNA interference inhibited VEGF-induced aortic ring sprouting, suggesting that anti-RPL29 strategies might have anti-angiogenic potential. Overall, our results identify that loss or depletion of RPL29 can reduce angiogenesis in vivo and ex vivo.

PTTG induces EMT through integrin αVß3-focal adhesion kinase signaling in lung cancer cells.

Pituitary tumor transforming gene (PTTG) is a well-studied oncogene for its role in tumorigenesis and serves as a marker of malignancy in several cancer types including lung. In the present study, we defined the role of PTTG in actin cytoskeleton remodeling, cell migration and induction of epithelial mesenchymal transition (EMT) through the regulation of integrin α(V)ß(3)-FAK (focal adhesion kinase) signaling pathway. Overexpression of PTTG through an adenovirus vector resulted in a significant increase in the expression of integrins α(V) and ß(3), a process that was reversed with the downregulation of PTTG expression through the use of an adenovirus expressing PTTG-specific small interfering RNA (siRNA). Western blot analysis of cells infected with adenovirus PTTG cDNA resulted in increased FAK and enhanced expression of adhesion complex molecules paxillin, metavincullin, and talin. Furthermore, downstream signaling genes Rac1, RhoA, Cdc42 and DOCK180 showed upregulation upon PTTG overexpression. This process was dependent on integrin α(V), as blockage by antagonist echistatin (RGD peptide) or α(V)-specific siRNA resulted in a decrease in FAK and subsequent adhesion molecules. Actin cytoskeleton disruption was detected as a result of integrin-FAK signaling by PTTG as well as enhanced cell motility. Taken together, our results suggest for the first time an important role of PTTG in regulation of integrins α(V) and ß(3) and adhesion-complex proteins leading to induction of EMT.

West Nile virus entry requires cholesterol-rich membrane microdomains and is independent of alphavbeta3 integrin.

West Nile virus (WNV) has been the leading cause of viral encephalitis in the United States since 1999. The endocytic processes involved in the internalization of infectious WNV by various cell types are not well characterized, and the involvement of cholesterol-rich membrane microdomains, or lipid rafts, in the life cycle of WNV has not been investigated previously. In this study, we found that the depletion of cellular cholesterol levels by brief treatment with methyl-beta-cyclodextrin resulted in a 100-fold reduction of the titers of infectious WNV released into the culture supernatant, as well as a reduction in the number of WNV genome copies in the cholesterol-depleted cells. The addition of exogenous cholesterol to cholesterol-depleted cells reversed this effect. Cholesterol depletion postinfection did not affect WNV growth, suggesting that the effect occurs at the level of WNV entry. We also showed that while WNV entry did not require alphavbeta3 integrin and focal adhesion kinase, WNV particles failed to be internalized by cholesterol-depleted cells. Finally, we showed the colocalization of the WNV envelope protein and cholera toxin B, which is internalized in a lipid raft-dependent pathway, in microdomain clusters at the plasma membrane. These data suggest that WNV utilizes lipid rafts during initial stages of internalization and that the lipid rafts may contain a factor(s) that may enhance WNV endocytosis.

Upregulation of osteoclast alpha2beta1 integrin compensates for lack of alphavbeta3 vitronectin receptor in Iraqi-Jewish-type Glanzmann thrombasthenia.

Osteoclasts utilize alphavbeta3 integrin adhesion to bone matrix during bone resorption. We have generated osteoclasts from the peripheral blood of Iraqi-Jewish patients with Glanzmann thrombasthenia (GT) who are completely deficient in beta3 integrin and exhibit a haemorrhagic diathesis resulting from the absence of platelet alphaIIbbeta3. We show that, in contrast to osteoclasts generated from normal subjects or patients with alphaIIb integrin deficiency, GT osteoclasts lack alphavbeta3. These osteoclasts exhibited a two- to fourfold increase in alpha2 and beta1 integrin expression, whereas other alphav integrins, including alphavbeta5, were not significantly affected. An accompanying decrease in bone resorption was observed, with 44% and 59% declines in pit number and depth, respectively, and resorption lacunae showed abnormal morphology on scanning electron microscopy. However, osteoclasts from GT developed in similar numbers to controls and exhibited an otherwise 'normal' phenotype. We conclude that the observed rise in alpha2beta1 expression compensates for the chronic genetic deficiency of alphavbeta3 in osteoclasts from patients with GT and is sufficient to enable bone resorption to proceed, albeit to a submaximal extent. This explains why Iraqi-Jewish patients with GT do not have osteopetrosis.