Srinivasan Shanmugarajan

Microarray Profile of Gene Expression During Osteoclast Differentiation in Modelled Microgravity

Microgravity (µXg) leads to a 10–15% loss of bone mass in astronauts during space flight. Osteoclast (OCL) is the multinucleated bone‐resorbing cell. In this study, we used the NASA developed ground‐based rotating wall vessel bioreactor (RWV), rotary cell culture system (RCCS) to simulate µXg conditions and demonstrated a significant increase (2‐fold) in osteoclastogenesis compared to normal gravity control (Xg). Gene expression profiling of RAW 264.7 OCL progenitor cells in modelled µXg by Agilent microarray analysis revealed significantly increased expression of critical molecules such as cytokines/growth factors, proteases and signalling proteins, which play an important role in enhanced OCL differentiation/function. Transcription factors such as c‐Jun, MITF and CREB implicated in OCL differentiation are upregulated; however no significant change in the levels of NFATc1 expression in preosteoclast cells subjected to modelled µXg. We also identified high‐level expression of calcium‐binding protein, S100A8 (calcium‐binding protein molecule A8/calgranulin A) in preosteoclast cells under µXg. Furthermore, modelled µXg stimulated RAW 264.7 cells showed elevated cytosolic calcium (Ca2+) levels/oscillations compared to Xg cells. siRNA knock‐down of S100A8 expression in RAW 264.7 cells resulted in a significant decrease in modelled µXg stimulated OCL differentiation. We also identified elevated levels of phospho‐CREB in preosteoclast cells subjected to modelled µXg compared to Xg. Thus, modelled µXg regulated gene expression profiling in preosteoclast cells provide new insights into molecular mechanisms and therapeutic targets of enhanced OCL differentiation/activation to prevent bone loss and fracture risk in astronauts during space flight missions. J. Cell. Biochem. 111: 1179–1187, 2010.

Bone loss in survival motor neuron (Smn−/−SMN2) genetic mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is characterized by degenerating lower motor neurons and an increased incidence of congenital bone fractures. Survival motor neuron (SMN) levels are significantly reduced due to deletions/mutations in the telomeric SMN1 gene in these patients. We utilized the Smn−/− SMN2 mouse model of SMA to determine the functional role for SMN in bone remodelling. µCT analysis of lumber vertebrae, tibia and femur bones from SMA mice revealed an osteoporotic bone phenotype. Histological analysis demonstrated a thin porous cortex of cortical bone and thin trabeculae at the proximal end of the growth plate in the vertebrae of SMA mice compared to wild‐type mice. Histochemical staining of the vertebrae showed the presence of abundant activated osteoclasts on the sparse trabeculae and on the endosteal surface of the thin cortex in SMA mice. Histomorphometric analysis of vertebrae from SMA mice showed an increased number of osteoclasts. Serum TRAcP5b and urinary NTx levels were elevated, consistent with increased bone resorption in these mice. SMA mice showed a significant decrease in the levels of osteoblast differentiation markers, osteocalcin, osteopontin and osterix mRNA expression; however, there were no change in the levels of alkaline phosphatase expression compared to WT mice. SMA mouse bone marrow cultures revealed an increased rate of osteoclast formation (54%) and bone resorption capacity (46%) compared to WT mice. Pre‐osteoclast cells from SMA mice showed constitutive up‐regulation of RANK receptor signalling molecules critical for osteoclast differentiation. Our results implicate SMN function in bone remodelling and skeletal pathogenesis in SMA. Understanding basic mechanisms of SMN action in bone remodelling may uncover new therapeutic targets for preventing bone loss/fracture risk in SMA. Copyright

Congenital Bone Fractures in Spinal Muscular Atrophy: Functional Role for SMN Protein in Bone Remodeling

Spinal muscular atrophy is the second most common fatal childhood disorder. Core clinical features include muscle weakness caused by degenerating lower motor neurons and a high incidence of bone fractures and hypercalcemia. Fractures further compromise quality of life by progression of joint contractures or additional loss of motor function. Recent observations suggest that bone disease in spinal muscular atrophy may not be attributed entirely to lower motor neuron degeneration. The presence of the spinal muscular atrophy disease-determining survival motor neuron gene (SMN), SMN expression, and differential splicing in bone-resorbing osteoclasts was recently discovered. Its ubiquitous expression and the differential expression of splice variants suggest that SMN has specific roles in bone cell function. SMN protein also interacts with osteoclast stimulatory factor. Mouse models of human spinal muscular atrophy disease suggest a potential role of SMN protein in skeletal development. Dual energy x-ray absorptiometry analysis demonstrated a substantial decrease in total bone area and poorly developed caudal vertebra in the mouse model. These mice also had pelvic bone fractures. Studies delineating SMN signaling mechanisms and gene transcription in a cell-specific manner will provide important molecular insights into the pathogenesis of bone disease in children with spinal muscular atrophy. Moreover, understanding bone remodeling in spinal muscular atrophy may lead to novel therapeutic approaches to enhance skeletal health and quality of life. This article reviews the skeletal complications associated with spinal muscular atrophy and describes a functional role for SMN protein in osteoclast development and bone resorption activity.

Mutant p62P392L Stimulation of Osteoclast Differentiation in Paget's Disease of Bone

Pagets disease of the bone (PDB) is an autosomal dominant trait with genetic heterogeneity, characterized by abnormal osteoclastogenesis. Sequestosome 1 (p62) is a scaffold protein that plays an important role in receptor activator of nuclear factor κB (RANK) signaling essential for osteoclast (OCL) differentiation. p62P392L mutation in the ubiquitin-associated (UBA) domain is widely associated with PDB; however, the mechanisms by which p62P392L stimulate OCL differentiation in PDB are not completely understood. Deubiquitinating enzyme cylindromatosis (CYLD) has been shown to negatively regulate RANK ligand-RANK signaling essential for OCL differentiation. Here, we report that CYLD binds with the p62 wild-type (p62WT), non-UBA mutant (p62A381V) but not with the UBA mutant (p62P392L) in OCL progenitor cells. Also, p62P392L induces expression of c-Fos (2.8-fold) and nuclear factor of activated T cells c1 (6.0-fold) transcription factors critical for OCL differentiation. Furthermore, p62P392L expression results in accumulation of polyubiquitinated TNF receptor-associated factor (TRAF)6 and elevated levels of phospho-IκB during OCL differentiation. Retroviral transduction of p62P392L/CYLD short hairpin RNA significantly increased TRAP positive multinucleated OCL formation/bone resorption activity in mouse bone marrow cultures. Thus, the p62P392L mutation abolished CYLD interaction and enhanced OCL development/bone resorption activity in PDB.

Role of CXC chemokine ligand 13 in oral squamous cell carcinoma associated osteolysis in athymic mice

Oral squamous cell carcinomas (OSCC) are malignant tumors with a potent activity of local bone invasion; however, the molecular mechanisms of tumor osteolysis are unclear. In this study, we identified high level expression of chemokine ligand, CXCL13 and RANK ligand (RANKL) in OSCC cells (SCC1, SCC12 and SCC14a). OSCC cell‐conditioned media (20%) induced osteoclast differentiation which was inhibited by OPG in peripheral blood monocyte cultures indicating that OSCC cells produce soluble RANKL. Recombinant hCXCL13 (10 ng/ml) significantly enhanced RANKL‐stimulated osteoclast differentiation in these cultures. Trans‐well migration assay identified that CXCL13 induces chemotaxis of peripheral blood monocytes in vitro which was inhibited by addition of anti‐CXCR5 receptor antibody. Zymogram analysis of conditioned media from OSCC cells revealed matrix metalloproteinase‐9 (MMP‐9) activity. Interestingly, CXCL13 treatment to OSCC cells induced CXCR5 and MMP‐9 expression suggesting an autocrine regulatory function in OSCC cells. To examine the OSCC tumor cell bone invasion/osteolysis, we established an in vivo model for OSCC by subcutaneous injection of OSCC cells onto the surface of calvaria in NCr‐nu/nu athymic mice, which developed tumors in 4–5 weeks. μCT analysis revealed numerous osteolytic lesions in calvaria from OSCC tumor‐bearing mice. Histochemical staining of calvarial sections from these mice revealed a significant increase in the numbers of TRAP‐positive osteoclasts at the tumor‐bone interface. Immunohistochemical analysis confirmed CXCL13 and MMP‐9 expression in tumor cells. Thus, our data implicate a functional role for CXCL13 in bone invasion and may be a potential therapeutic target to prevent osteolysis associated with OSCC tumors in vivo.

Factors that affect postnatal bone growth retardation in the twitcher murine model of Krabbe disease

Krabbe disease is an inherited lysosomal disorder in which galactosylsphingosine (psychosine) accumulates mainly in the central nervous system. To gain insight into the possible mechanism(s) that may be participating in the inhibition of the postnatal somatic growth described in the animal model of this disease (twitcher mouse, twi), we studied their femora. This study reports that twi femora are smaller than of those of wild type (wt), and present with abnormality of marrow cellularity, bone deposition (osteoblastic function), and osteoclastic activity. Furthermore, lipidomic analysis indicates altered sphingolipid homeostasis, but without significant changes in the levels of sphingolipid-derived intermediates of cell death (ceramide) or the levels of the osteoclast-osteoblast coupling factor (sphingosine-1-phosphate). However, there was significant accumulation of psychosine in the femora of adult twi animals as compared to wt, without induction of tumor necrosis factor-alpha or interleukin-6. Analysis of insulin-like growth factor-1 (IGF-1) plasma levels, a liver secreted hormone known to play a role in bone growth, indicated a drastic reduction in twi animals when compared to wt. To identify the cause of the decrease, we examined the IGF-1 mRNA expression and protein levels in the liver. The results indicated a significant reduction of IGF-1 mRNA as well as protein levels in the liver from twi as compared to wt littermates. Our data suggest that a combination of endogenous (psychosine) and endocrine (IGF-1) factors play a role in the inhibition of postnatal bone growth in twi mice; and further suggest that derangements of liver function may be contributing, at least in part, to this alteration.

Transgenic mice with OIP-1/hSca overexpression targeted to the osteoclast lineage develop an osteopetrosis bone phenotype†

Regulatory mechanisms operative in bone‐resorbing osteoclasts are complex. We previously defined the Ly‐6 gene family member OIP‐1/hSca as an inhibitor of osteoclastogenesis in vitro; however, a role in skeletal development is unknown. In this study, we developed transgenic mice with OIP‐1/hSca expression targeted to the osteoclast lineage that develop an osteopetrotic bone phenotype. Humeri from OIP‐1 mice showed a significant increase in bone mineral density and bone mineral content. µCT analysis showed increased trabecular thickness and bone volume. OIP‐1 mice have dense sclerotic cortical bone with absence of spongiosa and inadequate formation of marrow spaces compared to wild‐type mice. Moreover, complete inhibition of osteoclasts and marrow cavities in calvaria suggests defective bone resorption in these mice. OIP‐1 mouse bone marrow cultures demonstrated a significant decrease (41%) in osteoclast progenitors and inhibition (39%) of osteoclast differentiation/bone resorption. Western blot analysis further demonstrated suppression of TRAF‐2, c‐Fos, p‐c‐Jun, and NFATc1 levels in RANKL‐stimulated osteoclast precursors derived from OIP‐1 mice. Therefore, OIP‐1 is an important physiological inhibitor of osteoclastogenesis and may have therapeutic value against bone loss in vivo. Copyright

Osteoclast inhibitory peptide‐1 (OIP‐1) inhibits measles virus nucleocapsid protein stimulated osteoclast formation/activity

Pagets disease (PD) of bone is characterized by increased activity of large abnormal osteoclasts (OCLs) which contain paramyxoviral nuclear and cytoplasmic inclusions. MVNP gene expression has been shown to induce pagetic phenotype in OCLs. We previously characterized the osteoclast inhibitory peptide‐1 (OIP‐1/hSca) which inhibits OCL formation/bone resorption. OIP‐1 is a glycophosphatidylinositol (GPI)‐linked membrane protein containing a 79 amino acid extra cellular peptide and a 32 amino acid carboxy terminal GPI‐linked peptide (c‐peptide) which is critical for OCL inhibition. In this study, we demonstrate that OIP‐1 c‐peptide significantly decreased (43%) osteoclast differentiation of peripheral blood mononuclear cells from patients with PD. Also, OIP‐1 treatment to normal human bone marrow mononuclear cells transduced with the MVNP inhibited (41%) osteoclast precursor (CFU‐GM) growth in methyl‐cellulose cultures. We further tested if OIP‐1 overexpression in the OCL lineage in transgenic mice inhibits MVNP stimulated OCL formation. MVNP transduction and RANKL stimulation of OIP‐1 mouse bone marrow cells showed a significant decrease (43%) in OCL formation and inhibition (38%) of bone resorption area compared to wild‐type mice. Western blot analysis identified that OIP‐1 decreased (3.5‐fold) MVNP induced TRAF2 expression during OCL differentiation. MVNP or OIP‐1 expression did not affect TRAF6 levels. Furthermore, OIP‐1 expression resulted in a significant inhibition of MVNP stimulated ASK1, Rac1, c‐Fos, p‐JNK, and NFATc1 expression during OCL differentiation. These results suggest that OIP‐1 inhibits MVNP induced pagetic OCL formation/activity through suppression of RANK signaling. Thus, OIP‐1 may have therapeutic utility against excess bone resorption in patients with PD. J. Cell. Biochem. 104: 1500–1508, 2008.

IL-12 Stimulates the Osteoclast Inhibitory Peptide-1 (OIP-1/hSca) Gene Expression in CD4+ T Cells

Immune cell products such as interferon (IFN)‐γ and interleukin (IL)‐12 are potent inhibitors of osteoclast formation. We previously characterized the human osteoclast inhibitory peptide‐1 (OIP‐1/hSca), a Ly‐6 gene family member and showed IFN‐γ modulation of OIP‐1 expression in bone marrow cells. Whether, IL‐12 regulates OIP‐1 expression in the bone microenvironment is unclear. Real‐time PCR analysis revealed that IL‐12 treatment significantly enhanced OIP‐1 mRNA expression in human bone marrow mononuclear cells. Because IL‐12 induces IFN‐γ production by T cells, we tested whether IFN‐γ participates in IL‐12 stimulation of OIP‐1 gene expression in these cells. IL‐12 treatment in the presence of IFN‐γ neutralizing antibody significantly increased OIP‐1 mRNA expression, suggesting that IL‐12 directly regulates OIP‐1 gene expression. Interestingly, real‐time PCR analysis demonstrated that IL‐12 induces OIP‐1 expression (3.2‐fold) in CD4+ T cells; however, there was no significant change in CD8+ T cells. Also, IL‐12 (10 ng/ml) treatment of Jurkat cells transfected with OIP‐1 gene (−1 to −1,988 bp) promoter‐luciferase reporter plasmid demonstrated a 5‐fold and 2.7‐fold increase in OIP‐1 gene promoter activity in the presence and absence of antibody against IFN‐γ, respectively. We showed that STAT‐1,3 inhibitors treatment significantly decreased IL‐12 stimulated OIP‐1 promoter activity. Chromatin immunoprecipitation (ChIP) assay confirmed STAT‐3, but not STAT‐1 binding to the OIP‐1 gene promoter in response to IL‐12 stimulation. These results suggest that IL‐12 stimulates the OIP‐1 gene expression through STAT‐3 activation in CD4+ T cells. J. Cell. Biochem. 107: 104–111, 2009.

NIP45 negatively regulates RANK ligand induced osteoclast differentiation.

Receptor activator of NF‐κB ligand (RANKL)‐RANK receptor signaling to induce NFATc1 transcription factor is critical for osteoclast differentiation and bone resorption. RANK adaptor proteins, tumor necrosis factor receptor‐associated factors (TRAFs) play an essential role in RANKL signaling. Evidence indicates that NIP45 (NFAT interacting protein) binds with TRAFs and NFATc2. We therefore hypothesized that NIP45 regulates RANKL induced osteoclast differentiation. In this study, we demonstrate that RANKL treatment down regulates NIP45 expression in mouse bone marrow derived pre‐osteoclast cells. Lentiviral (pGIPZ) mediated shRNA knock‐down of NIP45 expression in RANKL stimulated pre‐osteoclast cells resulted in increased levels of NFATc1, NFATc2, and TRAF6 but not TRAF2 expression compared to control shRNA transduced cells. Also, NIP45 suppression elevated p‐IκB‐α levels and NF‐κB‐luciferase reporter activity. Confocal microscopy demonstrated NIP45 colocalized with TRAF6 in the cytosol of osteoclast progenitor cells. In contrast, RANKL stimulation induced NIP45 nuclear translocation and colocalization with NFATc2 in these cells. Coimmuneprecipitation assay demonstrated NIP45 binding with NFATc2 but not NFATc1. We further show that shRNA knock‐down of NIP45 expression in pre‐osteoclast cells significantly increased RANKL induced osteoclast differentiation and bone resorption activity. Taken together, our results indicate that RANKL signaling down regulates NIP45 expression and that NIP45 is a negative regulator of osteoclast differentiation. J. Cell. Biochem. 113: 1274–1281, 2012.