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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periodic restraint) as evidenced by corticosteroid-mediated atrophy of the spleen and/or thymus (Wang et al., 2007 and unpublished).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 PublicationsFujita N, Fujita S, Okada Y, Fujita K, Kitano A, Yamanaka O, Miyamoto T, Kon S, Uede T, Rittling SR, Denhardt DT, Saika S. (2009) Impaired angiogenic response in the cornea of mice lacking osteopontin. Invest Ophthalmol Vis Sci. Sep 9. [Epub ahead of print] Matsui Y, Iwasaki N, Kon S, Takahashi D, Morimoto J, Matsui Y, Denhardt DT, Rittling S, Minami A, Uede T. (2009) Accelerated development of aging-associated and instability-induced osteoarthritis in osteopontin-deficient mice. Arthritis Rheum. 60(8):2362-71. Sullivan J, Blair L, Alnajar A, Aziz T, Ng CY, Chipitsyna G, Gong Q, Witkiewicz A, Weber GF, Denhardt DT, Yeo CJ, Arafat HA. (2009) Expression of a prometastatic splice variant of osteopontin, OPNC, in human pancreatic ductal adenocarcinoma. Surgery. 146(2):232-40. Grassinger J, Haylock DN, Storan MJ, Haines GO, Williams B, Whitty GA, Vinson AR, Be CL, Li S, Sorensen ES, Tam PP, Denhardt DT, Sheppard D, Choong PF, Nilsson SK. (2009) Thrombin cleaved osteopontin regulates hemopoietic stem and progenitor cell functions through interactions with {alpha}9{beta}1 and {alpha}4{beta}1 integrins. Blood. 114(1):49-59. Chipitsyna G, Gong Q, Anandanadesan R, Alnajar A, Batra SK, Wittel UA, Cullen DM, Akhter MP, Denhardt DT, Yeo CJ, Arafat HA. (2009) Induction of osteopontin expression by nicotine and cigarette smoke in the pancreas and pancreatic ductal adenocarcinoma cells. Int J Cancer. 125(2):276-285. Maeno Y, Shinzato M, Nagashima S, Rittling SR, Denhardt DT, Uede T, Taniguchi K. (2009) Effect of osteopontin on diarrhea duration and innate immunity in suckling mice infected with a murine rotavirus. Viral Immunol. 22(2):139-44. Wang KX, Shi YF, Ron Y, Kazanecki CC, Denhardt DT. (2009) Plasma osteopontin modulates chronic restraint stress-induced thymus atrophy by regulating stress hormones: inhibition by an anti-osteopontin monoclonal antibody. J Immunol. 182(4):2485-91. Wang KX, Denhardt DT. (2008) Osteopontin: Role in immune regulation and stress responses. Cytokine Growth Factor Rev. 19(5-6):333-45. Diao H, Iwabuchi K, Li L, Onoe K, Van Kaer L, Kon S, Saito Y, Morimoto J, Denhardt DT, Rittling S, Uede T. (2008) Osteopontin regulates development and function of invariant natural killer T cells. Proc Natl Acad Sci U S A. 105(41):15884-9. Sato I, Yamamoto N, Rittling SR, Denhardt DT, Hino M, Morimoro J, Sakai F, Fujie A, Uede T. (2008) Osteopontin is dispensable for protection against high load systemic fungal infection. Int Immunopharmacol. 8(10):1441-8. Borges K, Gearing M, Rittling S, Sorensen ES, Kotloski R, Denhardt DT, Dingledine R. (2008) Characterization of osteopontin expression and function after status epilepticus. Epilepsia. 49(10):1675-85. Ono N, Nakashima K, Rittling SR, Schipani E, Hayata T, Soma K, Denhardt DT, Kronenberg HM, Ezura Y, Noda M. (2008) Osteopontin negatively regulates parathyroid hormone receptor signaling in osteoblasts. J Biol Chem. 283(28):19400-9. Miyazaki K, Okada Y, Yamanaka O, Kitano A, Ikeda K, Kon S, Uede T, Rittling SR, Denhardt DT, Kao WW, Saika S. (2008) Corneal wound healing in an osteopontin-deficient mouse. Invest Ophthalmol Vis Sci. 49(4):1367-75. Christensen 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. |
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