Interaction of Pyk2 and PTP-PEST with leupaxin in prostate cancer cells.

We have identified the presence of leupaxin (LPXN), which belongs to the paxillin extended family of focal adhesion-associated adaptor proteins, in prostate cancer cells. Previous studies have demonstrated that LPXN is a component of the podosomal signaling complex found in osteoclasts, where LPXN was found to associate with the protein tyrosine kinases Pyk2 and c-Src and the cytosolic protein tyrosine phosphatase-proline-, glutamate-, serine-, and threonine-rich sequence (PTP-PEST). In the current study, LPXN was detectable as a 50-kDa protein in PC-3 cells, a bone-derived metastatic prostate cancer cell line. In PC-3 cells, LPXN was also found to associate with Pyk2, c-Src, and PTP-PEST. A siRNA-mediated inhibition of LPXN resulted in decreased in vitro PC-3 cell migration. A recombinant adenoviral-mediated overexpression of LPXN resulted in an increased association of Pyk2 with LPXN, whereas a similar adenoviral-mediated overexpression of PTP-PEST resulted in decreased association of Pyk2 and c-Src with LPXN. The overexpression of LPXN in PC-3 cells resulted in increased migration, as assessed by in vitro Transwell migration assays. On the contrary, the overexpression of PTP-PEST in PC-3 cells resulted in decreased migration. The overexpression of LPXN resulted in increased activity of Rho GTPase, which was decreased in PTP-PEST-overexpressing cells. The increase in Rho GTPase activity following overexpression of LPXN was inhibited in the presence of Y27632, a selective inhibitor of Rho GTPase. In conclusion, our data demonstrate that LPXN forms a signaling complex with Pyk2, c-Src, and PTP-PEST to regulate migration of prostate cancer cells.

Association of leupaxin with Src in osteoclasts.

Leupaxin (LPXN), which belongs to the paxillin extended family of adaptor proteins, was previously identified as a component of the sealing zone in osteoclasts. LPXN was found to associate with several podosomal proteins, such as the protein tyrosine kinase Pyk2, the protein-tyrosine phosphatase-PEST (PTP-PEST), actin-binding proteins, and regulators of actin cytoskeletal reorganization. It was previously demonstrated that inhibition of LPXN expression resulted in reduced osteoclast-mediated resorption. In the current study, overexpression of LPXN in murine osteoclasts resulted in both enhanced resorptive activity and cell adhesion, as assessed by in vitro resorption assays. The overexpression of LPXN resulted in an increased association of Pyk2 with LPXN. In an attempt to determine an additional biochemical basis for the observed phenomenon in increased osteoclast activity, a coimmunoprecipitation screen for additional binding partners revealed that Src, a protein tyrosine kinase that is critical to both podosome formation and osteoclast function, was also associated with LPXN. After exposure to the pro-inflammatory and osteoclastogenic cytokine TNF-alpha, there was an increase in the level of Src that coimmunoprecipitated with LPXN. Our data indicate that association of the scaffold protein LPXN with Src adds further complexity to the organization of the podosomal signaling complex in osteoclasts.

Overexpression of the ZIP1 zinc transporter induces an osteogenic phenotype in mesenchymal stem cells.

Zinc is an essential trace element that is involved in diverse metabolic and signaling pathways. Zinc deficiency is associated with retardation of bone growth. Previous in vitro studies have suggested a direct effect of zinc on both the proliferation and differentiation of osteoblast-like cells. However, the mechanisms for uptake of zinc into osteoblasts have not been examined in detail. Several families of zinc transporters have previously been characterized in mammalian cells; such transporters function in the uptake, intracellular sequestration or efflux of zinc. In the current study, we examined zinc transport in osteoprogenitor cells and have attempted to define a functional role for a zinc transport mechanism in osteogenic differentiation. We identified at least two zinc transporters in both human mesenchymal stem cells (MSCs) and in osteoblastic cells--the ubiquitous zinc transporter, ZIP1, and LIV-1, which was previously characterized as a protein that is expressed in breast cancer cells. The subcellular localization of both these zinc transporters suggested distribution in both the plasma membrane and also diffusely in the cytoplasm. During the differentiation process of pluripotent MSCs into osteoblast-like cells, both zinc uptake and expression of the ZIP1 protein were increased. An adenoviral-mediated overexpression of ZIP1 in MSCs resulted in Alizarin-red-positive mineralization and also increased expression of specific osteoblast-associated markers, such as alkaline phosphatase, and of several osteoblast differentiation genes, including osteopontin, Cbfa1/Runx2, promyelocytic leukemia zinc finger and bone sialoprotein. An siRNA-mediated reduction of ZIP1 protein expression in MSCs caused decreased zinc uptake and inhibition of osteoblastic differentiation under osteogenic culture conditions. Finally, following overexpression of ZIP1 in MSCs, cDNA microarray analysis revealed differential regulation of several genes associated with the proliferation of osteoprogenitor cells and osteoblast differentiation. In conclusion, these studies provide important insights into the role of a plasma membrane zinc transporter in the initiation of an osteogenic lineage from MSCs.

Expression of the zinc transporter ZIP1 in osteoclasts.

Zinc has been previously demonstrated to be a potent inhibitor of osteoclastogenesis and osteoclast function. The mechanisms for cellular uptake of zinc into osteoclasts have not been characterized. We have corroborated previous studies on the reduction of osteoclastogenesis in the presence of extracellular zinc. We demonstrate that osteoclasts express a ubiquitous plasma membrane zinc transporter, namely ZIP1, which was diffusely distributed throughout the cytoplasm. Following an adenoviral-mediated overexpression of ZIP1 in murine osteoclasts, ZIP1 was predominantly colocalized with actin at the sealing zone and significantly inhibited osteoclast function, as assessed by resorptive activity. Finally, overexpression of ZIP1 negatively impacted NF-kappaB binding activity, as assessed by electrophoretic mobility shift assays. In conclusion, these data both corroborate previous studies on regulation of osteoclast formation and activity by zinc and reveal the presence of a zinc uptake mechanism that exerts an important effect on osteoclast activity.

Leupaxin is a critical adaptor protein in the adhesion zone of the osteoclast.

Leupaxin is a cytoskeleton adaptor protein that was first identified in human macrophages and was found to share homology with the focal adhesion protein, paxillin. Leupaxin possesses several protein-binding domains that have been implicated in targeting proteins such as focal adhesion kinase (pp125FAK) to focal adhesions. Leupaxin can be detected in monocytes and osteoclasts, both cells of hematopoietic origin. We have identified leupaxin to be a component of the osteoclast podosomal signaling complex. We have found that leupaxin in murine osteoclasts is associated with both PYK2 and pp125FAK in the osteoclast. Treatment of osteoclasts with TNF-alpha and soluble osteopontin were found to stimulate tyrosine phosphorylation of both leupaxin and leupaxin-associated PYK2. Leupaxin was found to co-immunoprecipitate with the protein tyrosine phosphatase PTP-PEST. The cellular distribution of leupaxin, PYK2, and protein tyrosine phosphorylation-PEST co-localized at or near the osteoclast podosomal complex. Leupaxin was also found to associate with the ARF-GTPase-activating protein, paxillin kinase linker p95PKL, thereby providing a link to regulators of cytoskeletal dynamics in the osteoclast. Overexpression of leupaxin by transduction into osteoclasts evoked numerous cytoplasmic projections at the leading edge of the cell, resembling a motile phenotype. Finally, in vitro inhibition of leupaxin expression in the osteoclast led to a decrease in resorptive capacity. Our data suggest that leupaxin may be a critical nucleating component of the osteoclast podosomal signaling complex.

Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions.

We previously demonstrated that inhibition of Na-dependent phosphate (P(i)) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/P(i) cotransporters in the osteoclast plasma membrane provide P(i) for ATP synthesis and that the osteoclast may utilize part of the P(i) released from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/P(i) cotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/P(i) cotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/P(i) cotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/P(i) cotransporters in the osteoclast.