Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-02-27
2004-09-14
Shukla, Ram R. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06790639
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to novel osteoregulin polypeptides, which play a role in regulating bone homeostasis, adiposity, and the calcification of atherosclerotic plaques. The invention also features screening assays to identify modulators of osteoregulin activity as well as methods of treating mammals for diseases or disorders associated with osteoregulin activity.
BACKGROUND OF THE INVENTION
Bone is a highly dynamic tissue that undergoes continual processes of remodeling and modeling (Parfitt, J. Cellular Biochemistry 55: 273-86, 1994). In the growing skeleton, the amount of mineralized bone formed exceeds the amount lost through resorption, whereas in the mature adult, bone loss and bone formation are equivalent, thereby preserving the integrity of the skeleton. Under certain conditions such as aging, postmenopausal estrogen deficiency, or prolonged steroid treatment, the amount of bone formed is not sufficient to compensate for the quantity lost by resorption. Over time, this imbalance results in reduced bone mass and compromises the structural integrity of the skeleton.
Bone remodeling is a very complex process of tightly coordinated action by the bone resorbing osteoclasts and the bone forming osteoblasts. Osteoblasts are derived from a mesenchymal cell lineage and are responsible for the formation of new bone matrix in their differentiated state (Wlodarski, Clinical Orthopaedics & Related Research 276: 93, 1990). In addition, factors produced by osteoblasts regulate the formation of osteoclasts and osteoctastic bone resorbing activity in response to endocrine signals such as parathyroid hormone and vitamin D. It has been postulated that the bone loss associated with aging is a result of a defect in the osteoblast cell lineage (Rodriguez et al., J. of Cellular Biochemistry 75: 414-423, 1999; Erdmann et al., Mechanisms of Aging & Development 110: 73-85, 1999; Roholl et al., J. of Bone & Mineral Research 9: 355-66, 1994; Katzburg et al., Bone 25: 667-673, 1999). Either the mesenchymal precursor population is insufficient or has lost the capacity to proliferate and differentiate into sufficient numbers of functioning osteoblasts.
Osteoblasts progress through a 3 stage process of differentiation: proliferation, matrix maturation, and mineralization (Aubin, Journal of Cellular Biochemistry 72: 396-410, 1999; Stein and Lian, Endocrine Reviews 14: 424-42, 1993; Malaval et al., J. of Cellular Biochemistry 74: 616-27, 1999). During this differentiation process, a well characterized temporal and spatial expression pattern of extracellular bone matrix proteins and other genes occurs (Malaval et al., J. of Cellular Biochemistry 74: 616-27, 1999; Owen et al., J. of Cellular Physiology 143: 420-30, 1990; Ingram et al., J. of Bone & Mineral Research 8: 1019-29, 1993). The bone matrix is composed primarily of Type I collagen which forms the extracellular structural component for the deposition of mineral forming hydroxyapatite. Osteoblasts also secrete non-collagenous proteins into the extracellular matrix (Robey, Connective Tissue Research 35: 131-6, 1996; Boskey, Connective Tissue Research 35: 357-63, 1996). The non-collagenous proteins include proteoglycans, sulfated glycoproteins, highly phosphorylated RGD-motif proteins, and proteins modified to contain gla amino acid residues. Examples include biglycan, osteonectin, bone sialoprotein, osteopontin, and osteocalcin. The exact function of many of these proteins have not yet been delineated although most evidence supports their role in promoting mineralization events (Robey, Connective Tissue Research 35: 131-6, 1996). An exception is the gla-peptide, osteocalcin, which has been shown to be a negative regulator of bone formation by gene knockout technology (Ducy et al.; Nature 382: 448-52, 1996).
The progression of osteoblast differentiation has been modeled in cell culture using primary calvarial cells or bone marrow cells. Bone marrow contains pluripotent stem cells of the adipocytic, osteoblastic, fibroblastic, and hematopoetic cell lineage (Owen et al., J. of Cellular Physiology 143: 420-30, 1990; Beresford, J. of Cell Science 102: 341-51, 1992: Herbertson and Aubin, Bone 21: 491-500, 1997). Bone marrow from rats, mice, and humans has been shown to contain osteoprogenitor cells that proliferate and can be induced to differentiate into osteoblastic cells. Rat and human cultures require a differentiation agent such as dexamethasone whereas mouse-derived cells can differentiate in the absence of dexamethasone (Rickard et al., J. of Bone & Mineral Research 11: 312-24, 1996; Maniatopoulos et al., Cell & Tissue Research 254: 317-30, 1988; Chen et al., Endocrinology 112: 1739-45, 1983). In vitro differentiated osteoblasts display the capacity to secrete noncollagenous proteins into the extracellular matrix in a temporally regulated manner which may indicate a regulatory function for each of these proteins in the mineralization process (Yao et al., J. of Bone & Mineral Research 9: 231-40, 1994). Furthermore, differentiated bone marrow cultures have the capacity to facilitate the deposition of matrix and the formation of hydroxyapatite mineral when grown in the presence of a phosphate source such as &bgr;-glycerophosphate. These properties have made the differentiation of bone marrow cells a useful model to investigate the mechanisms of bone remodeling and osteoblast function.
Osteoporosis accounts for approximately 700,000 fractures per year in the United States alone, and osteoporotic fractures are linked to significant death and is morbidity in the aged population. Therefore, there is a clear need to further understand the process of bone remodeling, both in normal and pathological states, in order to develop therapeutic agents to prevent, reduce, or reverse bone loss associated with osteoporosis or other bone-related disorders.
SUMMARY OF THE INVENTION
We sequenced a novel cDNA transcript expressed specifically in rat osteoblasts and osteocytes that encodes a 45 kDa polypeptide; and we have also identified the mouse and human forms. Our characterization revealed the protein to be a secreted, RGD motif containing protein with a limited homology to dmp1, an extracellular matrix protein present in bone and teeth (Roholl et al., J. of Bone & Mineral Research 9: 355-66, 1994; Katzburg et al., Bone 25: 667-673, 1999). Thus, we have designated this mammalian protein “osteoregulin.” Further studies of osteoregulin expression patterns and function (as further described in the detailed description) have confirmed that osteoregulin plays an important role in controlling bone homeostasis, adipose regulation, and the calcification of atherosclerotic plaques.
The invention features novel osteoregulin polypeptides, the nucleotide sequences that encode them, expression vectors containing these osteoregulin sequences, and transgenic hosts which have been genetically modified to express the osteoregulins of the invention.
Another feature of the invention is non-human mammals and animal cells that have been genetically-modified to disrupt one or both copies of an endogenous osteoregulin gene. Studies of genetically-modified mice that are homozygous or heterozygous for the osteoregulin gene disruption demonstrate that the absence or reduction of osteoregulin gene expression results in increased bone mass, increased bone mineralization, increased bone formation, and an increase in adiposity in females. These phenotypes indicate that osteoregulin functions as a negative regulator of bone mass/density and adiposity. This role in bone homeostasis is further supported by the significant expression of osteoregulin in bone tissue as well as osteoregulin's similarities to other proteins that play a role in regulating bone function.
Given the discovered function of osteoregulins as negative regulators of bone formation, bone density, bone mineralization, as well as its role in adiposity and plaque calcification, the present invention also features screening assays to identify agents that modulate osteoregulin activity or gene expressi
Brown Thomas A.
De Wet Jeffrey R.
Gowen Lori C.
Hames Lynn M.
Benson Gregg C.
Pfizer Inc.
Raymer Gregory P.
Richardson Peter C.
Shukla Ram R.
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