Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-05
2001-09-04
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007800, C435S455000
Reexamination Certificate
active
06284464
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of transcriptional regulation. More specifically, the present invention relates to inhibition of binding of hox and homeodomain-containing proteins and uses thereof.
2. Description of the Related Art
The bone morphogenetic proteins (BMPs), a subfamily of TGF&bgr;, are potent growth factors that regulate embryonic development, vertebral patterning and mesenchymal cell differentiation (1, 2). BMP-2/4, identified as bone inductive growth factors, are important signaling molecules during development of the skeleton in vertebrates (1, 3, 4). Central to the bone morphogenetic protein signaling pathway is the Smad1 protein, which translocates into the nucleus to regulate gene transcription upon direct phosphorylation by bone morphogenetic protein receptors (5-7).
Growth factors in the TGF&bgr; superfamily have been implicated in various processes during vertebrate embryonic development. The TGF&bgr; action on induction and patterning of mesoderm and skeletal development has been studied intensely. In particular, the TGF&bgr; related molecules, BMP-2/4 induces skeletal patterning, growth of limb buds and skeletal cell differentiation. Hox and homeodomain containing transcription factors are also involved in the same process, and have been suggested as a downstream regulation of BMP-4 to mediate its effects. However, there has been little progress in understanding how hox proteins function in embryonic development. Although hox proteins are DNA binding proteins, very little is known about their natural DNA response elements and their role in transcription.
In vertebrates, there are 39 Hox homeobox-containing transcription factor genes, organized into four separate chromosome clusters, which play critical roles in the process and patterning of vertebrate embryonic development (8,18). These 39 genes are subdivided into 13 paralogous groups on the basis of duplication of an ancestral homeobox cluster during evolution, sequence similarity and position within the cluster (9). Each paralog group has been demonstrated to be responsible for morphogenesis of a particular embryonic domain or structure (8). There are three members in Hox paralog group VIII, Hoxb-8, Hoxc-8 and Hoxd-8 (9). Hox genes are required during vertebrate limb bud development, and particularly, Hoxb-8 was suggested to be a transcription factor involved in activating the Sonic hedgehog gene, which is the key mediator in limb development (10,11). Furthermore, Northern blot analysis shows that Hoxc-8 is expressed during human embryo development at high levels in spinal cord, backbone and limbs and at a lower level in heart (12). BMP-2/4 activates expression of Hox genes, induces osteoblast differentiation and controls patterning across the anteroposterior (a-p) axis of developing limb (13).
The prior art is deficient in methods for stimulating osteoblast differentiation and bone formation. The prior art is also deficient in methods of regulating transcription via the Hox proteins and/or homeobox-containing proteins. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The bone morphogenetic protein-2 (BMP-2) was identified as a bone inductive growth factor, involved in inducing osteoblast differentiation. Central to the bone morphogenetic protein signaling pathway is the Smad1 protein, which translocates into the nucleus to activate osteoblast-specific gene transcription upon direct phosphorylation by bone morphogenetic protein receptors. The present invention identifies a specific interaction of Smad1 with Hox and homeodomain-containing proteins, which often act as transcriptional repressors, in which the binding of Smad1 to Hoxc-8 inhibits the recognition and binding of Hoxc-8 to its DNA binding site in a dose-dependent manner. This specific interaction between Smad1 and Hoxc-8 can be used as a target to inhibit the binding of Hoxc-8 to its DNA binding sites, thereby inducing osteoblast differentiation and preventing osteoporosis. The interaction of Smad1 with other Hox proteins or homeobox-containing proteins may also be used to regulate other diseases, such as cancer or cardiovascular disease.
In an embodiment of the present invention, there is provided a method of stimulating bone formation in an individual, comprising the step of: inducing an interaction between Smad1 and a homeobox-containing transcription factor. Preferably, this interaction induces a BMP-responsive gene which encodes a bone matrix protein. This induction results in osteoblast and/or chondroblast differentiation, which in turn, stimulates bone formation.
In another embodiment of the present invention, there is provided a method of inducing gene(s) encoding bone matrix proteins, comprising the step of: inducing an interaction between Smad1 and a homeobox-containing transcription factor in which the interaction results in an induction of gene(s) encoding bone matrix proteins. Specifically, there is provided a method of inducing a gene encoding osteopontin, comprising the steps of: inducing an interaction between Smad1 and Hoxc-8. Preferably, this interaction results in removal of the transcriptional repression and induction of the gene encoding osteopontin.
In still yet another embodiment of the present invention, there is provided a method of screening for a compound that stimulates bone formation, comprising the steps of: contacting a cell with a compound; and determining the ability of the compound to inhibit binding of Hoxc-8 to a gene. This inhibition of binding results in induction of the gene which indicates that the compound stimulates bone formation.
In yet another embodiment of the present invention, there is provided a method of regulating disease in an individual, comprising the step of: inhibiting the binding of a homeobox-containing transcription factor to a gene involved in regulating disease, wherein the inhibition removes transcriptional repression of the gene by the homeobox-containing protein and results in the induction of those genes involved in regulating disease.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
REFERENCES:
patent: 5693615 (1997-12-01), Stone
Langer, Drug Delivery and Targeting, Nature 392, suppl. p 5-10, Apr. 30, 1998.*
Anderson, Human Gene Therapy, Nature 392, suppl. p25-30, Apr. 30, 1998.
Cao Xu
Shi Xingming
Yang Xiangli
Adler Benjamin Aaron
Schwartzman Robert A.
UAB Research Foundation
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