AlphaV-integrins mediate the mechanoprotective action of osteopontin in podocytes

Increased mechanical load in podocytes due to glomerular hypertension is one of the important factors leading to podocyte damage and chronic kidney disease. In previous studies, we have shown that mechanical stretch increases osteopontin (OPN) expression in podocytes and that exogenous OPN is mechanoprotective via facilitating cytoskeletal reorganization of podocytes. In the present study, we asked whether the mechanoprotective effect of OPN in podocytes is mediated through specific integrins and whether endogenous OPN of podocytes is required for mechanoprotection. Conditionally immortalized mouse podocytes and primary podocytes (PP) from OPN-/- and OPN+/+ mice were used. Cyclic biaxial mechanical stretch (0.5 Hz, 7% linear strain) was applied for up to 3 days. Stretch-induced cell loss was ～30% higher in OPN-/- PP compared with OPN+/+ PP. Increased cell loss of OPN-/- PP was rescued by OPN coating. Analysis of integrin expression by RT-PCR, application of RGD and SLAYGLR peptides and anti-integrin antibodies, small-interfering RNA knockdown of integrins, and application of kinase inhibitors identified αV-integrins (αVβ1, αVβ3, and αVβ5) to mediate the mechano-protective effect of OPN in podocytes involving focal adhesion kinase, Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Our results demonstrate that endogenous OPN of podocytes plays a nonredundant role in podocyte adaptation to mechanical stretch, and that OPN signaling via α(V)-integrins may represent a relevant therapeutical target in podocytes.     

Unloading-induced bone loss was suppressed in gold-thioglucose treated mice

Loss of mechanical stress causes bone loss. However, the mechanisms underlying the unloading-induced bone loss are largely unknown. Here, we examined the effects of gold-thioglucose (GTG) treatment, which destroys ventromedial hypothalamus (VMH), on unloading-induced bone loss. Unloading reduced bone volume in control (saline-treated) mice. Treatment with GTG-reduced bone mass and in these GTG-treated mice, unloading-induced reduction in bone mass levels was not observed. Unloading reduced the levels of bone formation rate (BFR) and mineral apposition rate (MAR). GTG treatment also reduced these parameters and under this condition, unloading did not further reduce the levels of BFR and MAR. Unloading increased the levels of osteoclast number (Oc.N/BS) and osteoclast surface (Oc.S/BS). GTG treatment did not alter the basal levels of these bone resorption parameters. In contrast to control, GTG treatment suppressed unloading-induced increase in the levels of Oc.N/BS and Oc.S/BS. Unloading reduced the levels of mRNA expression of the genes encoding osteocalcin, type I collagen and Cbfa1 in bone. In contrast, GTG treatment suppressed such unloading-induced reduction of mRNA expression. Unloading also enhanced the levels of fat mass in bone marrow and mRNA expression of the genes encoding PPARgamma2, C/EBPalpha, and C/EBPbeta in bone. In GTG-treated mice, unloading did not increase fat mass and the levels of fat-related mRNA expression. These results indicated that GTG treatment suppressed unloading-induced alteration in bone loss.     

Osteopontin modulates CD44-dependent chemotaxis of peritoneal macrophages through G-protein-coupled receptors: evidence of a role for an intracellular form of osteopontin

Expression of osteopontin (OPN) by activated T-cells and macrophages is required for the development of cell-mediated inflammatory responses. Acting through integrin alpha(v)beta(3) and CD44 receptors, OPN can promote chemoattraction and pro-inflammatory cytokine expression by macrophages. In this study, we have used peritoneal macrophages from OPN-/, CD44-/-, and WT mice to study the relationship between OPN and CD44 in macrophage migration. Using confocal microscopy, we show that OPN co-distributes with CD44 inside macrophages at cell edges and in cell processes in a mutually dependent manner. The existence of an intracellular form of OPN is supported by pulse-chase studies in which a thrombin-sensitive, phosphorylated protein immunoprecipitated with OPN antibodies is retained inside macrophages. In OPN-/- and CD44-/- macrophages, the absence of CD44 and OPN, respectively, is associated with the formation of fewer cell processes, reduced cell fusion required to form functional multinucleated osteoclasts in the presence of CSF-1 and RANKL, and impaired chemotaxis. Whereas the chemotaxis of CD44-/- cells to various chemoattractants is almost completely abrogated, a differential effect is seen with the OPN-/- cells. Thus, OPN-/- cells migrate normally towards CSF-1 but not towards fMLP and MCP-1, which signal through G-protein coupled receptors (GPCRs). That the GPCR-mediated migration is dependent upon the level of cell-surface CD44 is indicated by the reduced cell-surface expression of CD44 in OPN-/- cells and a comparable impairment in the chemotaxis of CD44+/- cells. Although chemotaxis of OPN-/- cells could be rescued by an OPN substratum, or by addition of high levels of OPN in solution, no response is evident with physiological levels of OPN, indicating a requirement for the CD44-associated intracellular OPN in CD44 cell-surface expression. These studies indicate, therefore, that the level of cell surface CD44 is critical for GPCR-mediated chemotaxis by peritoneal macrophages and suggest that a novel intracellular form of OPN may modulate CD44 activities involved in these processes.     

Constitutively active parathyroid hormone receptor signaling in cells in osteoblastic lineage suppresses mechanical unloading-induced bone resorption

Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity.     

Simvastatin coating of TiO2 scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells

Bone tissue engineering requires an osteoconductive scaffold, multipotent cells with regenerative capacity and bioactive molecules. In this study we investigated the osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) on titanium dioxide (TiO₂) scaffold coated with alginate hydrogel containing various concentrations of simvastatin (SIM). The mRNA expression of osteoblast-related genes such as collagen type I alpha 1 (COL1A1), alkaline phosphatase (ALPL), osteopontin (SPP1), osteocalcin (BGLAP) and vascular endothelial growth factor A (VEGFA) was enhanced in hAD-MSCs cultured on scaffolds with SIM in comparison to scaffolds without SIM. Furthermore, the secretion of osteoprotegerin (OPG), vascular endothelial growth factor A (VEGFA), osteopontin (OPN) and osteocalcin (OC) to the cell culture medium was higher from hAD-MSCs cultured on scaffolds with SIM compared to scaffolds without SIM. The TiO₂ scaffold coated with alginate hydrogel containing SIM promote osteogenic differentiation of hAD-MSCs in vitro, and demonstrate feasibility as scaffold for hAD-MSC based bone tissue engineering.     

Rho-dependent Rho kinase activation increases CD44 surface expression and bone resorption in osteoclasts

Osteoclasts from osteopontin-deficient mice exhibit decreased CD44 surface expression [corrected]. Osteopontin (OPN)/alphavbeta3 generated Rho signaling pathway is required for the surface expression of CD44. In this work we show the Rho effector, Rho kinase (ROK-alpha), to be a potent activator of CD44 surface expression. ROK-alpha activation was associated with autophosphorylation, leading to its translocation to the plasma membrane, as well as its association with CD44. ROK-alpha promoted CD44 surface expression through phosphorylation of CD44 and ezrin-radixin-moesin (ERM) proteins and CD44.ERM.actin complex formation. Osteoclasts from OPN-/- mice exhibited an approximately 55-60% decrease in basal level ROK-alpha phosphorylation as compared with wild type osteoclasts. Furthermore, RhoVal-14 transduction was only partially effective in stimulating ROK-alpha/CD44 phosphorylation, as well as CD44 surface expression, in these osteoclasts. Studies on the inhibition of Rho by C3 transferase or ROK-alpha by the specific inhibitor, Y-27632, showed a decrease in the phosphorylation mediated by ROK-alpha and CD44 surface expression. Neutralizing antibodies to alphav, beta3, or CD44 inhibited the migration and bone resorption of wild type osteoclasts. However, only anti-alphav or -beta3 antibodies blocked OPN-induced phosphorylation of ROK-alpha, CD44, and the ERM proteins. Our results strongly suggest a role for ROK-alpha in alphavbeta3-mediated Rho signaling, which is required for the phosphorylation events and CD44 surface expression. The functional deficiencies in the Rho effector(s) because of the lack of OPN were associated with decreased CD44 surface expression and hypomotility in the OPN-/- osteoclasts. Finally, we find that cooperativity exists between alphavbeta3 and CD44 for osteoclast motility and bone resorption.     

Transient receptor potential vanilloid 4 deficiency suppresses unloading-induced bone loss

Mechanosensing is one of the crucial components of the biological events. In bone, as observed in unloading-induced osteoporosis in bed ridden patients, mechanical stress determines the levels of bone mass. Many molecules have been suggested to be involved in sensing mechanical stress in bone, while the full pathways for this event has not yet been identified. We examined the role of TRPV4 in unloading-induced bone loss. Hind limb unloading induced osteopenia in wild-type mice. In contrast, TRPV4 deficiency suppressed such unloading-induced bone loss. As underlying mechanism for such effects, TRPV4 deficiency suppressed unloading-induced reduction in the levels of mineral apposition rate and bone formation rate. In these mice, unloading-induced increase in the number of osteoclasts in the primary trabecular bone was suppressed by TRPV4 deficiency. Unloading-induced reduction in the longitudinal length of primary trabecular bone was also suppressed by TRPV4 deficiency. TRPV4 protein is expressed in both osteoblasts and osteoclasts. These results indicated that TRPV4 plays a critical role in unloading-induced bone loss.     

Osteopontin aggravates experimental autoimmune uveoretinitis in mice

Human endogenous uveitis is a common sight-threatening intraocular inflammatory disease and has been studied extensively using a murine model of experimental autoimmune uveoretinitis (EAU). It is possibly mediated by Th1 immune responses. In the present study, we investigated the role of osteopontin (OPN), a protein with pleiotropic functions that contributes to the development of Th1 cell-mediated immunity. Accompanying EAU progression, OPN was elevated in wild-type (WT) mice that had been immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide 1-20. OPN-deficient (OPN-/-) mice showed milder EAU progression in clinical and histopathological scores compared with those of WT mice. The T cells from hIRBP-immunized OPN-/- mice exhibited reduced Ag-specific proliferation and proinflammatory cytokine (TNF-alpha and IFN-gamma) production compared with those of WT T cells. When hIRBP-immunized WT mice were administered M5 Ab reacting to SLAYGLR sequence, a cryptic binding site to integrins within OPN, EAU development was significantly ameliorated. T cells from hIRBP-immunized WT mice showed significantly reduced proliferative responses and proinflammatory cytokine production upon stimulation with hIRBP peptide in the presence of M5 Ab in the culture. Our present results demonstrate that OPN may represent a novel therapeutic target to control uveoretinitis.     

Inactivation of Pink1 gene in vivo sensitizes dopamine-producing neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and can be rescued by autosomal recessive Parkinson disease genes, Parkin or DJ-1

Mutations in the mitochondrial PTEN-induced kinase 1 (Pink1) gene have been linked to Parkinson disease (PD). Recent reports including our own indicated that ectopic Pink1 expression is protective against toxic insult in vitro, suggesting a potential role for endogenous Pink1 in mediating survival. However, the role of endogenous Pink1 in survival, particularly in vivo, is unclear. To address this critical question, we examined whether down-regulation of Pink1 affects dopaminergic neuron loss following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the adult mouse. Two model systems were utilized: virally delivered shRNA-mediated knockdown of Pink1 and germ line-deficient mice. In both instances, loss of Pink1 generated significant sensitivity to damage induced by systemic MPTP treatment. This sensitivity was associated with greater loss of dopaminergic neurons in the Substantia Nigra pars compacta and terminal dopamine fiber density in the striatum region. Importantly, we also show that viral mediated expression of two other recessive PD-linked familial genes, DJ-1 and Parkin, can protect dopaminergic neurons even in the absence of Pink1. This evidence not only provides strong evidence for the role of endogenous Pink1 in neuronal survival, but also supports a role of DJ-1 and Parkin acting parallel or downstream of endogenous Pink1 to mediate survival in a mammalian in vivo context.     

Mechanical stimulation of osteopontin mRNA expression and synthesis in bone cell cultures

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) promotes alkaline phosphatase and procollagen type I gene expression in calvarial bone cells. The bone matrix glycoprotein osteopontin (OPN) is considered to be important in bone matrix metabolism and cell-matrix interactions, but its role is unknown. Here we examined the effects of IHC (13 kPa) on OPN mRNA expression and synthesis in primary calvarial cell cultures and the osteoblast-like cell line MC3T3-E1. OPN mRNA expression declined during control culture of primary calvarial cells, but not MC3T3-E1 cells. IHC upregulated OPN mRNA expression in late released osteoblastic cell cultures, but not in early released osteoprogenitor-like cells. Also, in both proliferating and differentiating MC3T3-E1 cells, OPN mRNA expression and synthesis were enhanced by IHC, differentiating cells being more responsive than proliferating cells. These results suggest a role for OPN in the reaction of bone cells to mechanical stimuli. The severe loss of OPN expression in primary bone cells cultured without mechanical stimulation suggests that disuse conditions down-regulate the differentiated osteoblastic phenotype. J. Cell. Physiol. 170:174–181, 1997. © 1997 Wiley-Liss, Inc.     

Genome-wide screening of alpha-tocopherol sensitive genes in heart tissue from alpha-tocopherol transfer protein null mice (ATTP(-/-))

Alpha-tocopherol transfer protein (ATTP) null mice (ATTP(-/-)) have a systemic deficiency of alpha-tocopherol (AT). The heart AT levels of ATTP(-/-) are <10% of those in ATTP(+/+) mice. The genomic responses of heart to AT deficiency were determined in 3 months old male ATTP(-/-) mice and compared with their ATTP(+/+) littermate controls using Affymetrix 430A 2.0 high density oligonucleotide arrays. Differential analysis of approximately 13000 genes identified repression of genes related to immune system and activation of genes related to lipid metabolism and inflammation with no significant change in the expression of classical antioxidant genes (catalase, superoxide dismutase, glutathione peroxidase) in ATTP(-/-) as compared to ATTP(+/+) mice. The present data identifies novel classes of AT sensitive genes in heart tissue.     

Thrombin hydrolysis of human osteopontin is dependent on thrombin anion-binding exosites

The cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic alpha(4)beta(1)/alpha(9)beta(1) integrin-binding motif (SVVYGLR), thereby acting as a potent cytokine for cells bearing these activated integrins. We show that purified milk OPN is a substrate for thrombin with a k(cat)/K(m) value of 1.14 x 10(5) m(-1) s(-1). Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC(50) = 1.2 +/- 0.2 microm), unfractionated heparin (IC(50) = 56.6 +/- 8.4 microg/ml) and low molecular weight (5 kDa) heparin (IC(50) = 31.0 +/- 7.9 microg/ml), indicating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II). Using a thrombin mutant library, we mapped residues important for recognition and cleavage of OPN within ABE-I and ABE-II. A peptide (OPN-(162-197)) was designed spanning the OPN thrombin cleavage site and a hirudin-like C-terminal tail domain. Thrombin cleaved OPN-(162-197) with a specificity constant of k(cat)/K(m) = 1.64 x 10(4) m(-1) s(-1). Representative ABE-I mutants (K65A, H66A, R68A, Y71A, and R73A) showed greatly impaired cleavage, whereas the ABE-II mutants were unaffected, suggesting that ABE-I interacts principally with the hirudin-like OPN domain C-terminal and contiguous to the thrombin cleavage site. Debye-Hückel slopes for milk OPN (-4.1 +/- 1.0) and OPN-(162-197) (-2.4 +/- 0.2) suggest that electrostatic interactions play an important role in thrombin recognition and cleavage of OPN. Thus, OPN is a bona fide substrate for thrombin, and generation of thrombin-cleaved OPN with enhanced pro-inflammatory properties provides another molecular link between coagulation and inflammation.     

Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.     

Chorein deficiency leads to upregulation of gephyrin and GABA(A) receptor

Chorea-acanthocytosis (ChAc) is a hereditary neurodegenerative disorder caused by loss of function mutations in the VPS13A gene encoding chorein. Recently, using a gene-targeting technique to delete exons 60-61, we produced a ChAc-model mouse that corresponds to a human disease mutation. In this study, a comparative microarray analysis of gene expression in the striatum revealed an increased level of gephyrin gene expression in the ChAc-model mice compared with wild type mice. Since gephyrin is known as a GABA(A) receptor-anchoring protein, we compared the protein-level expression and localization of gephyrin and the GABA(A) receptor alpha1 (GABRA1) and gamma2 (GABRG2) subunits. Gephyrin and GABRG2 immunoreactivities in the striatum and hippocampus of the ChAc-model mice were significantly higher than those in the wild types. Our results suggest that chorein functional loss may lead to a compensatory upregulation of gephyrin and GABRG2 in the pathologic condition in ChAc.     

Molecular markers for identification of Stellantchasmus falcatus and a phylogenic study using the HAT-RAPD method

Stellantchasmus falcatus is a minute intestinal fluke in the family Heterophyidae. Metacercariae, the infective stage, were reported in a marine fish, mullet Liza subviridis, and a fresh water fish, Dermogenus pusillus, in Thailand. Adults were found in chicks, rats, cats, and humans. Morphological studies were done for comparing Stellantchasmus sp. worms found in 2 different fish hosts; their shapes and organ arrangements were very similar except for the prepharynx length. Therefore, the present study aimed to compare their DNA fingerprints using the HAT-RAPD method for both types of Stellantchasmus and several other related species. Ten arbitrarily selected primers (OPA-04, OPA-09, OPN-02, OPN-03, OPN-09, OPN-12, OPP-11, OPR-15, OPX-13, and OPAD-01) were used. It was found that OPA-09, OPN-03, and OPAD-01 were able to generate S. falcatus specific fragments in mullets which consisted of 200, 760, and 280 bp, respectively. In addition, the results of morphologic, DNA fingerprinting, and phylogenetic analyses strongly suggest that the fresh water and marine specimens of Stellantchamus may be different species.     

Osteoclast migration on phosphorylated osteopontin is regulated by endogenous tartrate-resistant acid phosphatase

Osteopontin (OPN) is a multifunctional protein implicated in cellular adhesion and migration. Phosphorylation has emerged as a post-translational modification important for certain biological activities of OPN. This study demonstrates that adhesion of isolated neonatal rat osteoclasts in vitro was augmented on bovine milk osteopontin (bmOPN) with post-translational modifications (PTMs) compared to human Escherichia-coli-derived recombinant OPN (hrOPN) without PTMs. The difference in adhesiveness between these OPN variants was more pronounced at low coating concentrations (≤ 10 μg/ml). Both OPN forms adhered exclusively using a β₃-integrin. Partial (≤50%) dephosphorylation by tartrate-resistant acid phosphatase (TRAP) in vitro reduced osteoclast attachment to bmOPN to the same level as to hrOPN, demonstrating the importance of specific phosphorylations in OPN-dependent osteoclast adhesion.The involvement of PTMs of OPN in migration of primary rat and mouse osteoclasts was assessed on culture dishes coated with the different OPN forms and then overlaid with gold particles. Here, osteoclasts exhibited haptotactic migration on bmOPN but did not migrate on hrOPN. The presence of neutralizing antibodies to TRAP inhibited migration on bmOPN. Moreover, migration of osteoclasts isolated from TRAP-overexpressing transgenic mice was augmented on bmOPN, but not on hrOPN or type I collagen.These data collectively provide evidence in favor of a role for endogenous TRAP in regulating osteoclast migration on post-translationally modified OPN. In a tissue context, modulation of the phosphorylation level of OPN by extracellular phosphatases, e.g., TRAP, could regulate the extent of degradation such as depth and area at each bone resorption site by triggering osteoclast detachment and facilitate subsequent migration on the bone surface.