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On the systems biology of osteopontin: Role in autoimmune disease, stress responses, bone remodeling and cancer metastasisThe 2007 year has seen our recent research culminate in three publications. Christian Kazanecki, who recently completed his PhD thesis, showed that two structurally different forms of OPN differ in how each signals cells. OPN produced by osteoblasts is much more highly modified (phosphorylated on serine) than OPN produced by ras-transformed fibroblasts (Kazanecki et al., 2007a; Christensen et al., 2007). FbOPN contains approximately four phosphate groups distributed over 17 potential phosphorylation sites, whereas ObOPN contains approximately 21 phosphate groups distributed over 27 sites. Five residues were O-glycosylated in both isoforms. The two proteins were compared in adhesion assays for their ability to support attachment of human MDA-MB-435 tumor cells and mouse ras-transformed 275-3-2 fibroblast cells. FbOPN mediated binding of the MDA-MD-435 cells with 50% higher efficiency than ObOPN. The opposite trend was observed for the mouse fibroblast line where ObOPN promoted approximately 30% higher binding efficiency than FbOPN. His research has suggested an interesting structural feature (a β-sheet fold) that permits the C-terminal end of the OPN molecule to regulate integrin binding at a central RGD sequence via a CD44 interaction (Kazanecki et al., 2007b). In another study, PhD student Kathryn Wang has shown that OPN confers on mice a sensitivity to certain forms of stress (hindlimb unloading, periodic restraint) as evidenced by corticosteroid-mediated atrophy of the spleen and/or thymus (Wang et al., 2007 and unpublished). We hypothesize that OPN is necessary for the stress-induced elevation of corticosterone expression; Henry Rutgers Honors student Jennifer Luo is investigating by RT-QPCR whether transcription of one or more genes involved in steroid synthesis is OPN-regulated. Our major goal in the immediate future is to identify and characterize an anti-OPN monoclonal antibody that is a functional inhibitor of OPN action in vivo. Such an antibody may be clinically useful in the treatment of autoimmune disease, cancer metastasis, bone resorption (osteoporosis), and pathological stress responses. We have successfully isolated several dozen new anti-OPN monoclonal antibodies some of which recognize post-translational modifications (PTMs), largely serine phosphorylations, in the protein. To identify the epitopes recognized by the different monoclonal antibodies we have employed two strategies. One utilizes peptides corresponding to conserved sequences in the OPN protein that are either phosphorylated or unphosphorylated. This allows us to determine whether a particular MAb is specific for that PTM. The second strategy employed two different forms of OPN, the highly post-translationally modified native OPN purified from human milk and the unmodified form synthesized in E. coli. The native form was labeled with the fluorescent tag Oregon Green and the recombinant form labeled with Texas Red. In the novel assay developed during this study by Masters student Josephine Cassella the two forms were mixed and allowed to react in solution with the monoclonal antibody under investigation. The antigen-antibody complexes were then captured on magnetic beads coupled to protein G (which binds the Fc portion of the IgG). After washing the beads, the adsorbed IgG was eluted at low pH, and the fluorescence at the two wavelengths determined. MAbs that selectively bound the native protein (relative to the modified protein) are presumed to be antibodies that recognize PTMs. This can be confirmed using the peptide assay mentioned above. This new method is important in that it detects the antibody-antigen reaction in solution rather than on a solid support, as occurs in the Western and ELISA assays. Hybridomas with the potential to produce monoclonal antibodies recognizing PTMs were prepared in two ways, both of which employed our OPN-null (knockout) mouse. (This is important because OPN is found in the plasma of normal mice, and such mice would be tolerant of the protein and unable to make anti-OPN antibodies very efficiently.) In the “standard” approach the mice were immunized with the highly modified human milk OPN. In the alternate approach mice were immunized with a set of five serine-phosphorylated peptides designed to mimic five regions in the OPN protein that are highly conserved between human, mouse, rat and bovine OPN species. Monoclonals derived from these immunizations are currently being tested as described above to determine their specificity. We are now in the process of identifying MAbs that are functional inhibitors of OPN, both in cell culture and in vivo, in the mouse. In cell culture, we have shown that exogenous OPN can inhibit the apoptotic response of human umbilical vein endothelial cells deprived of growth factors (Khan et al., 2005). In ongoing work, Dana Cifelli, a Masters student, is investigating the ability of selected MAbs to suppress the apoptosis-inhibiting action of OPN. In the mouse, Kathryn Wang is continuing her studies described above to determine whether any of our anti-OPN MAbs can block the ability of OPN to cause immune organ atrophy in mice exposed to stress. (We have recently established that it is OPN in the plasma that is necessary for the organ atrophy.) Should this be successful in identifying specific MAbs that inhibit this action of OPN, we would explore their usefulness as compounds that might be clinically useful in the treatment of autoimmune disease, cancer metastasis, certain forms of stress and/or osteoporosis. Selected PublicationsChristensen B, Kazanecki CC, Petersen TE, Rittling SR, Denhardt DT, Sørensen ES. (2007) Cell type-specific post-trans-lational modifications of mouse osteopontin are associated with different adhesive properties. J Biol Chem. 282:19463-72. Hashimoto M, Sun D, Rittling SR, Denhardt DT, Young W. (2007) Osteopontin-deficient mice exhibit less inflammation, greater tissue damage, and impaired locomotor recovery from spinal cord injury compared with wild-type controls.
J Neurosci. 2007 27:3603-11. Kazanecki CC, Uzwiak DJ, Denhardt DT. (2007) Control of osteopontin signaling and function by post-translational
phosphorylation and protein folding. J Cell Biochem. 102:912-924. Kitamura M, Iwabuchi K, Kitaichi N, Kon S, Kitamei H, Namba K, Yoshida K, Denhardt DT, Rittling SR, Ohno S, Uede T, Onoe K. (2007) Osteopontin aggravates experimental autoimmune uveoretinitis in mice. J Immunol. 178(10):6567-72. Ishijima M, Tsuji K, Rittling SR, Yamashita T, Kurosawa H, Denhardt DT, Nifuji A, Ezura Y, Noda M. (2007)Osteopontin is required for mechanical stress-dependent signals to bone marrow cells. J Endocrinol. 193(2):235-43. Kato N, Kitahara K, Rittling SR, Nakashima K, Denhardt DT, Kurosawa H, Ezura Y, Noda M. (2007) Osteopontin deficiency enhances anabolic action of EP4 agonist at a sub-optimal dose in bone. J Endocrinol. 193(1):171-82. Hashimoto M, Sun D, Rittling SR, Denhardt DT, Young W. (2007) Osteopontin-deficient mice exhibit less inflammation, greater tissue damage, and impaired locomotor recovery from spinal cord injury compared with wild-type controls. J Neurosci. 27(13):3603-11. Kavukcuoglu NB, Denhardt DT, Guzelsu N, Mann AB. (2007) Osteopontin deficiency and aging on nanomechanics of mouse bone. J Biomed Mater Res A. 83:136-44. Wung JK, Perry G, Kowalski A, Harris PL, Bishop GM, Trivedi MA, Johnson SC, Smith MA, Denhardt DT, Atwood CS. (2007) Increased expression of the remodeling- and tumorigenic-associated factor osteopontin in pyramidal neurons of the Alzheimer's disease brain. Curr Alzheimer Res. 4(1):67-72. Marsh BC, Kerr NC, Isles N, Denhardt DT, Wynick D. (2007) Osteopontin expression and function within the dorsal root ganglion. Neuroreport. 18(2):153-7. Saika S, Shirai K, Yamanaka O, Miyazaki K, Okada Y, Kitano A, Flanders KC, Kon S, Uede T, Kao WW, Rittling SR, Denhardt DT, Ohnishi Y. (2007) Loss of osteopontin perturbs the epithelial-mesenchymal transition in an injured mouse lens epithelium. Lab Invest. 87(2):130-8. Mori N, Majima T, Iwasaki N, Kon S, Miyakawa K, Kimura C, Tanaka K, Denhardt DT, Rittling S, Minami A, Uede T. (2007) The role of osteopontin in tendon tissue remodeling after denervation-induced mechanical stress deprivation. Matrix Biol. 26(1):42-53. Ishijima M, Ezura Y, Tsuji K, Rittling SR, Kurosawa H, Denhardt DT, Emi M, Nifuji A, Noda M.(2006) Osteopontin is associated with nuclear factor kappaB gene expression during tail-suspension-induced bone loss. Exp Cell Res. 312(16):3075-83. Da Silva AP, Pollett A, Rittling SR, Denhardt DT, Sodek J, Zohar R. (2006) Exacerbated tissue destruction in DSS-induced acute colitis of OPN-null mice is associated with downregulation of TNF-alpha expression and non-programmed cell death. J Cell Physiol. 208(3):629-39. Lai CF. Seshadri V. Huang K. Shao JS. Cai J. Vattikuti R. Schumacher A. Loewy AP. Denhardt DT. Rittling SR. Towler DA. (2006) An Osteopontin-NADPH oxidase Signaling cascade promotes pro-matrix Metalloproteinase 9 activation in Aortic Mesenchymal cells. Circ Res. 98:1479-89. Khan SA. Cook AC. Kappil M. Gunthert U. Chambers AF. Tuck AB. Denhardt DT. (2006) Enhanced cell surface CD44 variant (v6. v9) expression by osteopontin in breast cancer epithelial cells facilitates tumor cell migration: Novel post-transcriptional. post-translational regulation. Clin Exp Metastasis. 22:663-73. Schroeter M. Zickler P. Denhardt DT. Hartung HP. Jander S. (2006) Increased thalamic neurodegeneration following ischaemic cortical stroke in osteopontin-deficient mice. Brain. 129:1426-37. Maeno Y. Nakazawa S. Yamamoto N. Shinzato M. Nagashima S. Tanaka K. Sasaki J. Rittling SR. Denhardt DT. Uede T. Taniguchi K. (2006) Osteopontin participates in Th1-mediated host resistance against nonlethal malaria parasite Plasmodium chabaudi chabaudi infection in mice. Infect Immun. 74:2423-7. Koyama Y. Rittling SR. Tsuji K. Hino K. Salincarnboriboon R. Yano T. Taketani Y. Nifuji A. Denhardt DT. Noda M. (2006) Osteopontin Deficiency Suppresses High Phosphate Load-Induced Bone Loss via Specific Modulation of Osteoclasts. Endocrinology. 147:3040-9. Khan SA, Cook AC, Kappil M, Gunthert U, Chambers AF, Tuck AB, Denhardt DT. (2005) Enhanced cell surface CD44 variant (v6, v9) expression by osteopontin in breast cancer epithelial cells facilitates tumor cell migration: novel post-transcriptional, post-translational regulation. Clin Exp Metastasis. 22:663-73. Zhu Y. Denhardt DT. Cao H. Sutphin PD. Koong AC. Giaccia AJ. Le QT. (2005) Hypoxia upregulates osteopontin expression in NIH-3T3 cells via a Ras-activated enhancer.Oncogene. 43:6555-63. Kondo H. Nifuji A. Takeda S. Ezura Y. Rittling S. Denhardt DT. Nakashima K. Karsenty G. Noda M. (2005) Unloading induces osteoblastic cell suppression and osteoclastic cell activation to lead to bone loss via sympathetic nervous system. J Biol Chem. 34:30192-200. Nilsson SK. Johnston HM. Whitty GA. Williams B. Webb RJ. Denhardt DT. Bertoncello I. Bendall LJ. Simmons PJ. Haylock DN. (2005) Osteopontin. a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells. Blood. 106:1232-9. Contractor T. Babiarz B. Kowalski AJ. Rittling SR. Sorensen ES. Denhardt DT. (2005) Osteoclasts resorb protein-free mineral (Osteologic discs) efficiently in the absence of osteopontin. In Vivo. 19:335-41. Rollo EE. Hempson SJ. Bansal A. Tsao E. Habib I. Rittling SR. Denhardt DT. Mackow ER. Shaw RD. (2005) The cytokine osteopontin modulates the severity of rotavirus diarrhea. J Virol. 79:3509-16. Porter JF. Shen S. Denhardt DT (2004) Tissue Inhibitor of Metalloproteinase-1 stimulates proliferation of human cancer cells by inhibiting a metalloproteinase. Brit J Cancer. 90:463-70. Shapses SA. Cifuentes M. Speva L. Chowdhury H. Brittingham J. Boskey AL. Denhardt DT (2003) Osteopontin facilitates bone resorption. decreasing bone mineral crystallinity and content during calcium deficiency. Calcified Tissues Int. 73:86-92. Denhardt. D.T.. Mistretta. D.. Chambers. A.F.. Krishna. S.. Porter J.F.. Raghuram. S.. Rittling. S.R. (2003) Transcriptional regulation of the osteopontin promoter and the metastatic phenotype: Evidence for a Ras-activated enhancer in the human OPN promoter. Clin. Expt. Metastasis 20:77-84. Noda. M.. and Denhardt. D. T. (2002) Osteopontin. In Principles of Bone Biology. Edited by JP Bilezikian. LG Raisz. and GA Rodan 2nd ed. vol 1. pp239-250. Academic Press. Khan. S.. Lopez. CA. Zhang. J. Sørensen. E and Denhardt. DT (2002) PON inhibits apoptosis of endothlial cells deprived of growth factors. J Cellular Biochem 85:728-73. D'Alonzo RC. Kowalski AJ. Denhardt DT. Nickols GA. Partridge NC. (2002) Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells. J Biol Chem. 277:24788-98. Khan. S.. Lopez. C.A.. Zhang. J.. Sørensen. E.. and Denhardt. D.T. (2002) Osteopontin inhibits apoptosis of endothelial cells deprived of growth factors. J. Cellular Biochemistry 85:728-736.
Khan SA. Lopez-Chua CA. Zhang J. Fisher LW. Sorensen ES. Denhardt DT.
(2002) Soluble
osteopontin inhibits apoptosis of adherent endothelial cells deprived of
growth factors. Denhardt. D.T.. Giachelli. C.M.. and Rittling. S.R. (2001) Role of Osteopontin in Cellular Signaling and Toxic Injury. Ann. Rev. Pharmacology and Toxicology 41:721-749. Denhardt. D.T.. Noda. M.. O'Regan. A.E.. Pavlin. D.. and Berman. J.S. (2001) OPN: a means to cope with environmental insults: Regulation of inflammation. tissue remodeling and cell survival. J. Clin. Invest. 107:1055-1061. Denhardt. D.T.. Burger. E.H.. Kazanecki. C.. Krishna. S.. Semeins. C.M.. and Klein-Nulend. J. (2001) Osteopontin-deficient bone cells are defective in their ability to produce NO in response to pulsatile fluid flow. Biochem. Biophys. Res. Comm. 288:448-453. Chabas. D.. Baranzini. S.E.. Mitchell. D.. Bernard. C.C.A.. Rittling. S.R.. Denhardt. D.T.. Sobel. R.A.. Lock. C. Karpuj. M. Pedotti. R.. Heller. R. Oksenberg. J.R.. and Steinman. L. (2001) The influence of the proinflammatory cytokine. osteopontin. on autoimmune demyelinating disease. Science 294:1731-1735. Denhardt DT. Burger EH. Kazanecki C. Krishna S. Semeins CM. Klein-Nulend J. (2001) Osteopontin-deficient bone cells are defective in their ability to produce NO in response to pulsatile fluid flow. Biochem Biophys Res Commun. 288:448-53. Denhardt DT. Noda M. O'Regan AW. Pavlin D. Berman JS. (2001) Osteopontin as a means to cope with environmental insults: regulation of inflammation. tissue remodeling. and cell survival. J Clin Invest. 107:1055-61. Asou. Y.. Rittling. S.R.. Yoshitake. H.. Tsuji. K.. Shinomiya. K.. Nifuji. A.. Denhardt. D.T.. and Noda. M. (2001) Osteopontin facilitates angiogenesis. accumulation of osteoclasts. and resorption of ectopic bone. Endocrinology 142:1325-1332. Attur. M.G.. Dave. M.N.. Stuchin. S.. Kowalski. A.S.. Steiner. G.. Abramson. S.B.. Denhardt. D.T.. and Amin. A.R. (2001) Osteopontin: An intrinsic inhibitor of inflammation in cartilage. Arthritis and Rheumatism 44:578-584. Ishijimi. M.. Rittling. S.R.. Yamashita. T.. Tsuji. K.. Kurosawa. H.. Nifuji. A.. Denhardt. D.T.. and Noda. M. (2001) Enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of osteopontin. J. Exp. Med. 193:399-404. Nemoto. H.. Rittling. S.R.. Yoshitake. H.. Furuya. K.. Amagasa. T.. Tsuji. K.. Nifuji. A.. Denhardt. D.T.. Noda. M. (2001) Osteopontin deficiency reduces experimental tumor cell metastasis to bone and soft tissues. J. Bone Mineral Res. 16:652-659. Denhardt. D.T.. Giachelli. C.M.. Rittling. S.R. (2001) Role of osteopontin in cellular signaling and toxicant injury. Annu. Rev. Pharmacol. Toxicol. 41:721-749. Hampel. D.J.. Sansome. C.. Romanov. V.I.. Kowalski. A.J.. Denhardt. D.T.. and Goligorsky. M.S. (2001) Osteopontin traffic in stressed renal epithelial cells. Am. J. Physiol. 94:66-76 Ihara H. Denhardt DT. Furuya K. Yamashita T. Muguruma Y. Tsuji K. Hruska KA. Higashio Enomoto S. Nifuji A. Rittling SR. Noda M. (2001) Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin. J. Biol. Chem. 276:13065-71. Ashkar. S.. Weber. G.F.. Panoutsakopoulou. .V. Sanchirico. M.E.. Jansson. M.. Zawaideh. S.. Rittling. S.R.. Denhardt. D.T.. Glimcher. M.J.. Cantor. H. (2000) Eta -1 (osteopontin): an early component of type -1 (cell -mediated) immunity. Science 287. 86086 -4. Denhardt. D.T. (2000) On the paradoxical ability of TIMPs either to inhibit or to promote the development and progression of the malignant phenotype. In ""Inhibitors of Metalloproteinases in Development and Disease"". Pp 137-151. Edited by S.P. Hawkes. D.R. Edwards. and R. Khokha. Harwood Academic Publ. Lausanne. Switzerland. Tian. J.T.. Søørensen. E.S.. Butler. W.T.. Lopez. C.A.. Sy. M.-S.. and Denhardt. D.T. (2000) Osteopontin and Hyaluronic Acid Inhibit NO Synthesis by RAW264.7 Cells Stimulated with Inflammatory Mediators: Collagen Blocks Inhibition by Osteopontin. Cytokine 12:450-457. Bonvini. J.M.. Schatzmann. U.. Beck-Schimmer. B.. Sun. L.K.. Rittling. S.R.. Denhardt. D.T.. Hir. M.L.. and Wuthrich. R.P. (2000) Lack of an in vivo function of osteopontin in experimental anti-GBM nephritis. J. Am. Soc. Nephrol. 11:1647-1655. Wu Y. Denhardt DT. Rittling SR. (2000) Osteopontin is required for full expression of the transformed phenotype by the ras oncogene. Br J Cancer. 83:156-63. |
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