Mehran Amoui

Expression of a Structurally Unique Osteoclastic Protein-tyrosine Phosphatase Is Driven by an Alternative Intronic, Cell Type-specific Promoter

An osteoclastic protein-tyrosine phosphatase (PTP-oc), essential for osteoclast activity, shows sequence identity with the intracellular domain of GLEPP1, a renal receptor-like transmembrane PTP. PTP-oc has been assumed to be a truncated variant of GLEPP1, resulting from alternative splicing. However, the 5′-untranslated region sequence of PTP-oc mRNA contains 217 bp from an intron of GLEPP1. There are no splicing acceptor sites at the PTP-oc transcription site. The intronic sequence flanking the 5′ end of the PTP-oc transcription start site contains potential promoter elements essential for transcriptional initiation. To test the hypothesis that the PTP-oc gene has an alternative, tissue-specific, intronic promoter, the promoter activity of a 1.3-kb PCR fragment covering the 5′-flanking region of the PTP-oc gene was measured. The putative PTP-oc promoter fragment showed strong promoter activity in U937 cells. Mutation of the putative TATA box within the PTP-oc promoter abolished 60–90% of its promoter activity. The PTP-oc promoter fragment showed strong promoter activity in cells that express PTP-oc (U937 cells and RAW264.7 cells) but not in cells that do not express the enzyme (skin fibroblasts, TE85 cells, and HEK293 cells). These findings strongly support the conclusion that the 1.3-kb intronic fragment contains the tissue-specific, PTP-oc proximal promoter. Deletion and functional analyses indicate that the proximal 5′ sequence flanking the TATA box of the PTP-oc contains potential repressor elements. The removal of the putative repressor elements led to the apparent loss of tissue specificity. In summary, we conclude that an intronic promoter within the GLEPP1 gene drives the expression of the PTP-oc in a cell type-specific manner. This GLEPP1/PTP-oc gene system is one of the very few systems in which two important tissue-specific enzymes are derived from the same gene by the use of alternative intronic promoters.

Leptin Receptor (Lepr) Is a Negative Modulator of Bone Mechanosensitivity and Genetic Variations in Lepr May Contribute to the Differential Osteogenic Response to Mechanical Stimulation in the C57BL/6J and C3H/HeJ Pair of Mouse Strains

This study investigated the role of leptin receptor (Lepr) signaling in determining the bone mechanosensitivity and also evaluated whether differences in the Lepr signaling may contribute to the differential osteogenic response of the C57BL/6J (B6) and C3H/HeJ (C3H) pair of mouse strains to mechanical stimuli. This study shows that a loading strain of ∼2,500 μϵ, which was insufficient to produce a bone formation response in B6 mice, significantly increased bone formation parameters in leptin-deficient ob−/ob− mice and that a loading strain of ∼3,000 μϵ also yielded greater osteogenic responses in Lepr-deficient db−/db− mice than in wild-type littermates. In vitro, a 30-min steady shear stress increased [3H]thymidine incorporation and Erk1/2 phosphorylation in ob−/ob− osteoblasts and db−/db− osteoblasts much greater than those in corresponding wild-type osteoblasts. The siRNA-mediated suppression of Lepr expression in B6 osteoblasts enhanced (but in osteoblasts of C3H (the mouse strain with poor bone mechanosensitivity) restored) their anabolic responses to shear stress. The Lepr signaling (leptin-induced Jak2/Stat3 phosphorylation) in C3H osteoblasts was higher than that in B6 osteoblasts. One of the three single nucleotide polymorphisms in the C3H Lepr coding region yielded an I359V substitution near the leptin binding region, suggesting that genetic variation of Lepr may contribute to a dysfunctional Lepr signaling in C3H osteoblasts. In conclusion, Lepr signaling is a negative modulator of bone mechanosensitivity. Genetic variations in Lepr, which result in a dysfunctional Lepr signaling in C3H mice, may contribute to the poor osteogenic response to loading in C3H mice.

Targeted Transgenic Expression of an Osteoclastic Transmembrane Protein-tyrosine Phosphatase in Cells of Osteoclastic Lineage Increases Bone Resorption and Bone Loss in Male Young Adult Mice *

This study evaluated whether transgenic expression of PTP-oc (osteoclastic transmembrane protein-tyrosine phosphatase) in cells of the osteoclast lineage would affect bone resorption and bone density in young adult mice. Transgenic mice were generated with a transgenic construct using a tartrate-resistant acid phosphatase exon 1C promoter to drive expression of rabbit PTP-oc in osteoclastic cells. pQCT evaluation of femurs of young adult male progeny of three lines showed that transgenic mice had reduced bone volume and area, cortical and trabecular bone mineral content, and density. Histomorphometric analyses at secondary spongiosa of the femur and at metaphysis of the L4 vertebra confirmed that male transgenic mice had decreased trabecular surface, reduced percentage of trabecular area, decreased trabecular number, increased trabecular separation, and increased osteoclast number per bone surface length. Consistent with an increase in bone resorption, the serum C-telopeptide level was 25% higher in transgenic mice than in wild-type littermates. However, the bone phenotype was not readily observed in female young adult transgenic mice. This could in part be due to potential interactions between estrogen and PTP-oc signaling, since the bone loss phenotype was seen in young adult ovariectomized transgenic mice by microcomputed tomography analysis. In vitro, the average pit area per resorption pit created by marrow-derived transgenic osteoclasts was ∼50% greater than that created by wild-type osteoclasts. Transgenic osteoclasts showed a lower c-Src phosphotyrosine 527 level, greater c-Src kinase activity, and increased tyrosine phosphorylation of paxillin. In summary, this study provides compelling in vivo evidence that PTP-oc is a positive regulator of osteoclasts.

An osteoclastic protein-tyrosine phosphatase is a potential positive regulator of the c-Src protein-tyrosine kinase activity: A mediator of osteoclast activity

This study tested the hypothesis that an osteoclastic protein‐tyrosine phosphatase, PTP‐oc, enhances osteoclast activity through c‐Src activation. The effects of several resorption activators and inhibitors on PTP‐oc expression, resorption activity, and c‐Src activation were determined in rabbit osteoclasts. PTP‐oc expression was assayed with immunoblots and semi‐quantitative RT‐PCR. Osteoclastic activity was determined by the resorption pit assay; and c‐Src activation was monitored by P‐tyr527 (PY527) dephosphorylation, and in vitro kinase assay. Treatment of osteoclasts with PTH, PGE2, 1,25(OH)2D3, IL‐1, but not RANKL or IL‐6, significantly stimulated resorption activity, increased PTP‐oc mRNA and protein levels, and reduced c‐Src PY527 level with corresponding activation of c‐Src protein‐tyrosine kinase activity. The PTP‐oc antisense phosphorothioated oligo treatment blocked the basal and IL‐1α‐mediated, but not RANKL‐mediated, resorption activity of isolated osteoclasts. The antisense oligo treatment also significantly reduced the average depth of resorption pits created by rabbit osteoclasts under basal conditions. Calcitonin and alendondrate, significantly reduced resorption activity and PTP‐oc expression, and increased c‐Src PY527 with corresponding reduction in its PTK activity. The cellular PTP‐oc protein level correlated with the resorption activity. Among the various signaling proteins co‐immunoprecipitated with PTP‐oc, the resorption effectors caused corresponding changes in the tyrosyl phosphorylation level of only c‐Src. The GST–PTP‐oc fusion protein dephosphorylated PY‐527‐containing c‐Src peptide in time‐ and dose‐dependent manner in vitro. In summary, (1) PTP‐oc is regulated in part at transcriptional level, (2) upregulation of PTP‐oc in osteoclasts led to c‐Src activation, and (3) PY527 of c‐Src may be a cellular substrate of PTP‐oc. These findings are consistent with the hypothesis that PTP‐oc is a positive regulator of c‐Src in osteoclasts. J. Cell. Biochem. 97: 940–955, 2006.

Targeted Overexpression of Osteoactivin in Cells of Osteoclastic Lineage Promotes Osteoclastic Resorption and Bone Loss in Mice

This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ∼2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by μ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption.

EphA4 Receptor Is a Novel Negative Regulator of Osteoclast Activity

Of the ephrin (Eph) receptors, mature osteoclasts express predominantly EphA4. This study sought to determine if EphA4 has a regulatory role in osteoclasts. Treatment of RAW/C4 cells with Epha4 small interfering RNAs (siRNAs) increased average size, Ctsk mRNA expression level, and bone resorption activity of the derived osteoclast‐like cells. Activation of the EphA4 signaling in osteoclast precursors with EfnA4‐fc chimeric protein reduced cell size and resorption activity of the derived osteoclasts. Homozygous Epha4 null mice had substantially less trabecular bone in femur and vertebra compared to wild‐type controls. The bone loss was due to a decrease in trabecular number and an increase in trabecular spacing, but not to an increase in osteoclast‐lined bone surface or an increase in the number of osteoclasts on bone surface. Dynamic histomorphometry and serum biomarker analyses indicate that bone formation in Epha4 null mice was reduced slightly but not significantly. Osteoclasts of Epha4 null mice were also larger, expressed higher levels of Mmp3 and Mmp9 mRNAs, and exhibited greater bone resorption activity than wild‐type osteoclasts in vitro. Deficient Epha4 expression had no effects on the total number of osteoclast formed in response to receptor activator of NF‐κB ligand nor on apoptosis of osteoclasts in vitro. It also did not affect the protein‐tyrosine phosphorylation status of its ligands, EfnB2, EfnA2, and EfnA4, in osteoclasts. Deficient Epha4 expression in Epha4 null osteoclasts activated the β3‐integrin signaling through reduced phosphorylation of the tyr‐747 residue, which led to increased binding of the stimulatory talin and reduced binding of the inhibitory Dok1 to β3‐integrin. This in turn activated Vav3 and the bone resorption activity of osteoclasts. In conclusion, we demonstrate for the first time that EphA4 is a potent negative regulator of osteoclastic activity, mediated in part through increased Dok1 binding to β3‐integrin via an increase in EphA4‐dependent tyr‐747 phosphorylation.

Targeted deletion of the osteoclast protein‐tyrosine phosphatase (PTP‐oc) promoter prevents RANKL‐mediated osteoclastic differentiation of RAW264.7 cells

An osteoclastic protein‐tyrosine phosphatase, PTP‐oc, shares the same gene with a renal PTP, Glepp1. This study demonstrated that targeted deletion of PTP‐oc promoter by homologous recombination in RAW264.7 cells completely abolished PTP‐oc expression without affecting Glepp1 expression. This strategy to inhibit PTP‐oc function has three advantages over commonly used gene knock down strategies (e.g., small interference RNA). This strategy: (1) yielded cells completely devoid of PTP‐oc, (2) had no off‐target gene silencing effects, and (3) did not affect Glepp1 expression. The inability of PTP‐oc‐deficient RAW264.7 cells to undergo RANKL‐mediated osteoclastic differentiation confirmed a regulatory role for PTP‐oc in RANKL‐mediated osteoclast differentiation.

An osteoclastic protein-tyrosine phosphatase regulates the β3-integrin, syk, and shp1 signaling through respective src-dependent phosphorylation in osteoclasts

This study utilized the glutathione transferase (GST) pull-down assay to identify novel substrates of an osteoclastic protein-tyrosine phosphatase, PTP-oc. Consistent with the previous findings that the phosphorylated tyr-527 (pY527) of Src is a substrate of PTP-oc, the major protein pulled down with the phosphatase-deficient (PD)-PTP-oc-GST trapping mutant in RAW264.7 cells was Src. The GST-PD-PTP-oc also pulled down pY-Syk and pY-β(3)-integrin, but not after PP2 pretreatment. However, PTP-oc transgenic osteoclasts or PTP-oc-overexpressing RAW264.7 cells had elevated, and not reduced, levels of pY525/526-Syk and pY759-β(3) integrin, and the PTP-oc siRNA treatment drastically reduced levels of pY525/526 Syk and pY759-β(3)-integrin in RAW264.7 cells. These findings are incompatible with the premise that they are substrates of PTP-oc. The PTP-oc-dependent increases in pY525/526-Syk and pY759-β(3)-integrin levels were completely blocked by PP2, indicating that these effects are secondary to PTP-oc-mediated activation of the Src protein-tyrosine kinase (PTK). Overexpression of PTP-oc increased, and siRNA-mediated suppression of PTP-oc reduced, pY160-Vav1, pY173-Vav3, and pY783-PLCγ levels, and Rac1 activation, which are downstream mediators of the ITAM/Syk signaling. Overexpression of PTP-oc also increased, and PTP-oc siRNA treatment decreased, the pY-Shp1 levels, which were blocked by PP2. Since Shp1 is a negative regulator of osteoclast activity and is a key mediator of the ITIM signaling, these findings suggest that PTP-oc is an upstream suppressor of the ITIM/Shp1 signaling through PTP-oc-induced Src-dependent Shp1 phosphorylation. In summary, PTP-oc plays a central regulatory role in the concerted regulation of the β(3)-integrin, the ITAM/Syk, and the ITIM/Shp1 signaling indirectly through activation of Src PTK.

An Osteoclastic Transmembrane Protein-Tyrosine Phosphatase Enhances Osteoclast Activity in Part by Dephosphorylating EphA4 in Osteoclasts.

We have previously shown that PTP‐oc is an enhancer of the functional activity of osteoclasts and that EphA4 is a suppressor. Here, we provide evidence that PTP‐oc enhances osteoclast activity in part through inactivation of EphA4 by dephosphorylating key phosphotyrosine (pY) residues of EphA4. We show that EphA4 was pulled down by the PTP‐oc trapping mutant but not by the wild‐type (WT) PTP‐oc and that transgenic overexpression of PTP‐oc in osteoclasts drastically decreased pY602 and pY779 residues of EphA4. Consistent with the previous findings that EphA4 deficiency increased pY173‐Vav3 level (Rac‐GTP exchange factor [GEF]) and enhanced bone resorption activity of osteoclasts, reintroduction of WT‐Epha4 in Epha4 null osteoclasts led to ∼50% reduction in the pY173‐Vav3 level and ∼2‐fold increase in bone resorption activity. Overexpression of Y779F‐Epha4 mutant in WT osteoclasts markedly increased in pY173‐Vav3 and reduced bone resorption activity, but overexpression of Y602F‐Epha4 mutant had no effect, suggesting that pY779 residue plays an important role in the EphA4‐mediated suppression of osteoclast activity. Deficient EphA4 in osteoclasts has been shown to up‐regulate Rac‐GTPase and down‐regulate Rho‐GTPase. PTP‐oc overexpression in osteoclasts also increased the GTP‐Rac level to 300% of controls, but decreased the GTP‐Rho level to ∼50% of controls. PTP‐oc overexpression or deficient Epha4 each also reduced pY87‐Ephexin level, which is a Rho GEF. Thus, PTP‐oc may differentially regulate Rac signaling versus Rho signaling through dephosphorylation of EphA4, which has shown to have opposing effects on Rac‐GTPase versus Rho‐GTPase through differential regulation of Vav3 versus Ephexin. J. Cell. Biochem. 116: 1785–1796, 2015.

Conditional Disruption of miR17∼92 in Osteoclasts Led to Activation of Osteoclasts and Loss of Trabecular Bone in Part Through Suppression of the miR17-Mediated Downregulation of Protein-Tyrosine Phosphatase-oc in Mice: miR17 INHIBITS OSTEOCLAST ACTIVATION THROUGH SUPPRESSION OF PTP-OC

This study sought to understand the regulation of an osteoclastic protein‐tyrosine phosphatase (PTP‐oc), a positive regulator of osteoclast activity. Our past studies suggested that PTP‐oc is regulated posttranscriptionally. The 3′‐untranslated region (UTR) of PTP‐oc mRNA contains a target site for miR17. During osteoclastic differentiation, there was an inverse relationship between the cellular levels of miR17 (expressed as one of the six cluster genes of miR17∼92) and PTP‐oc mRNA. Overexpression of pre‐miR17∼92 in mouse osteoclast precursors reduced PTP‐oc mRNA level and the size of the derived osteoclasts, whereas deletion of miR17∼92 or inhibition of miR17 resulted in the formation of larger osteoclasts containing more nuclei that expressed higher PTP‐oc mRNA levels and created larger resorption pits. Thus, PTP‐oc–mediated osteoclast activation is modulated in part by miR17∼92, particularly miR17. The miR17∼92 osteoclast conditional knockout (cKO) mutants, generated by breeding miR17∼92loxp/loxp mice with Ctsk‐Cre mice, had lower trabecular bone volume/total volume (Tb.BV/TV), trabecular bone mineral density (Tb.BMD), trabecular connectivity density (Tb.Conn‐Dens), trabecular number (Tb.N), and trabecular thickness (Tb.Th), but larger trabecular separation (Tb.Sp), and greater bone resorption without a change in bone formation compared to littermate controls. The cKO marrow‐derived osteoclasts were twice as large, contained twice as many nuclei, and produced twice as large resorption pits as osteoclasts of littermate controls. The expression of genes associated with osteoclast activation was increased in cKO osteoclasts, suggesting that deletion of miR17∼92 in osteoclasts promotes osteoclast activation. The cKO osteoblasts did not show differences in cellular miR17 level, alkaline phosphatase activity, and bone nodule formation ability. In conclusion, miR17‐92 negatively regulates the osteoclast activity, in part via the miR17‐mediated suppression of PTP‐oc in osteoclasts.