2000 — 2003 |
Frenkel, Baruch |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Chromatinized Genes in Osteoblasts @ University of Southern California
DESCRIPTION (Adapted from the Investigator's Abstract): Glucocorticoids (GC) are commonly used as anti-inflammatory and immunosuppressive drugs. The major adverse effect of long-term therapy is osteoporosis. Although the mechanisms are not well known, effects of GC on osteoblasts are thought to be a key component. The osteocalcin (OC) gene is a well-established useful model to study osteoblast-specific gene expression and GC are known to down-regulate osteocalcin transcription. The investigator has developed osteoblast cell lines that stably express osteocalcin promoter regions linked to a reporter gene. The stable cell lines down-regulate expression of the reporter gene in response to GC treatment. Importantly, identical constructs transiently expressed in these lines do not respond to GC. Therefore, the investigator proposes that osteocalcin and perhaps other osteoblast phenotypic genes may be initially in a chromatin-repressed state. These genes are de-repressed at specific times in development and their expression results in the commitment of the cell to the osteoblast differentiation pathway. The hypothesis asserts that GC act to prevent the de-repression and maintain osteoblast genes (eg osteocalcin) in a repressed state. The stably integrated osteocalcin promoter/reporter genes will serve as the model system to test this hypothesis. Chromatin modifications within the osteocalcin promoter and the effect of GC on these modifications will be examined by ChIP assays and endonuclease accessibility assays. The stably integrated promoter constructs will be used to identify DNA elements and transcription factors that mediate the suppressive effects of GC within the native chromatin context in osteoblast cell lines. Specific histone acetyltransferase activities (CBP and P300) will be examined to determine the effect of GC on the level or activity of specific HAT proteins.
|
1 |
2002 — 2003 |
Frenkel, Baruch |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Identification of Cbfa1 Targets in Osteoblasts @ University of Southern California
DESCRIPTION (provided by applicant): CBFA1 is a transcription factor with the most well established role in osteoblast differentiation and biomineralization. However, there are hardly any known CBFA1 target genes, which seem to play the anticipated critical role in promoting biomineralization. No study has been reported to date, describing an unbiased pursuit of CBFA1 target genes. This application proposes to clone new CBFA1 targets in osteoblasts, with the long-term goal of discovering genes playing critical roles in biomineralization. Traditional approaches to discover CBFA1 targets (e.g., differential display, microarrays) would compare mRNAs from cells with normal, low, or high levels of CBFA 1. However, such approaches often result in a long list of genes, for many of which the altered expression is secondary and of little importance to the transcription factor and to the biological process of interest. In addition, these approaches would normally entail over-expression of CBFA1 to supra-physiological levels, possibly leading to the identification of targets with limited biological significance. We have begun to develop a novel approach, by which CBFA1 target genes would be cloned based on their physical interaction with CBFA1 in living osteoblasts. Then CBFA1 targets will be isolated from a pool of genomic DNA fragments obtained by chromatin immunoprecipitation (ChIP) with CBFA1 antibodies. Our preliminary experiments show that, using the most optimal ChIP conditions, known CBFA1 targets are enriched by only 50-fold. By itself, this would be insufficient for isolating CBFA1 targets, due to vast excess of fragments that would non-specifically coprecipitate along with true CBFA1 targets. Towards establishing a method of purifying true CBFA1 targets, we first showed that efficient ChIP could be performed using restriction enzyme digestion instead of sonication for fragmentation of the chromatin. This will facilitate the concentration of CBFA1 target genes, each now represented by a unique size fragment, using polyacrylamide gel electrophoresis. Non-specifically precipitated, contaminating fragments will be spread along a much larger area of the gel. Identification of true CBFA1 targets will be further facilitated by segregating the ChiPped DNA into families of fragments based on the identity of nucleotides at the ends of each fragment. This step, achieved by amplification of the Chipped fragments with pairs of anchored primers, will further increase the intensity of bands representing true CBFA1 fragments over the background. Finally, the most intense bands will be eluted from the gel, cloned and sequenced. Genetically adjacent ORFs will be identified and their regulation by CBFA1 validated.
|
1 |
2005 — 2007 |
Frenkel, Baruch |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Chip Display of Runx2 Targets in Osteoblasts @ University of Southern California
DESCRIPTION (provided by applicant): The molecular mechanisms of mammalian tissue biomineralization are poorly understood. Ample evidence suggests a key role for the transcription factor Runx2 in both physiological and pathological calcification. However, there is little knowledge of Runx2 target genes that mediate its biological activities. We invented chromatin immunoprecipitation (ChIP) Display (CD), a method that allows the identification of target genes for transcription factors in an unbiased fashion. CD entails digestion of DNA obtained by ChIP with a restriction enzyme, followed by amplification and segregation of the precipitated DNA into thirty-six distinct families of fragments. This approach allows us to concentrate true targets on gels, while scattering the overwhelming background generated during ChIP. Preliminary CD experiments, presenting potential Runx2 targets by gel electrophoresis of Avall-digested ChIP, have already yielded four novel Runx2 targets. We propose three specific aims: 1) To complete the Runx2 CD with AvaII and to perform a similar CD using a different restriction enzyme, Tfil. We anticipate the identification of approximately one hundred Runx2 targets most highly occupied by Runx2 in living osteoblasts. We will compare the results of the AvaII and Tfil mediated CD analyses to assess the degree to which we are able to comprehensively discover Runx2 target genes. 2) To compare the results of Runx2 CD analyses to the results obtained by hybridization of Runx2 ChIP to CpG island microarrays. This will provide an independent measure of the power of CD to discover Runx2 target genes. 3) To test each potential Runx2 target from Aims 1 and 2 for: (i) occupancy by Runx2 using conventional ChIP assay with gene-specific primers; (ii) pattern of gene expression during osteoblast differentiation; and (iii) direct regulation by Runx2, using promoter-reporter assays for a focused group of Runx2 targets. The assembled repertoire of Runx2 target genes will serve the basis for future studies, in which selected candidates will be evaluated functionally for their role in osteoblast differentiation, bone formation and biomineralization.
|
1 |
2006 — 2010 |
Frenkel, Baruch |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Krox 20 and Bone Mass @ University of Southern California
DESCRIPTION (provided by applicant): Krox20 and Bone Mass. We propose to delineate the role of the transcription factor Krox20 in the regulation of osteoblast function and bone mass control. During the previous funding cycle of this project, we discovered that Krox20 expression and transcriptional activity were strongly inhibited by glucocorticoids (GCs), contributing to the repression of osteocalcin transcription in cultured osteoblasts. We propose to expand the project beyond the problem of GC-induced osteoporosis (GIO) and study the role of Krox20 in basic osteoblast biology in vivo and in vitro. Such a role is suggested by: (i) developmental arrest of trabecular bone formation in Krox20-/- mice;(ii) low trabecular bone mass and decreased bone formation rate in mature Krox20 mice;(iii) impaired mineralization in Krox20 and Krox20-/- osteoblast cultures;(iv) stimulation of Krox20 expression and activity by the Wnt signaling pathway;and (v) expression pattern in vivo and in vitro consistent with a role for Krox20 in osteoblast function. We will analyze as a function of age the skeletal phenotype of mice with insufficient and/or osteoblast-specific excess of Krox20. In vivo approaches will include microcomputed tomography (mu CT) as well as histological and serological analyses. The cellular and molecular targets of Krox20 will be determined analyzing osteoblast cultures in which Krox20 expression has been altered by either genetic manipulations or viral infection. The in vitro analyses will be performed at short intervals during the development of the osteoblast phenotype to identify the Krox20-sensitive differentiation stage. The specific roles of Krox20 will be determined with respect to cell cycle progression, apoptosis, development of biochemical markers and expression of genes that promote the osteoblast phenotype, including genes potentially regulated directly by Krox20 and indirectly by the Wnt signaling pathway. Finally, we will return to the investigation of GIO. We will assess the effect of GCs on Krox20 expression in vivo in the context of a detailed description (including mu CT and apoptosis) of GIO development in the mouse;and, we will determine whether treatment with a Krox20 virus or recombinant Wnt3A antagonize adverse effects of GCs in osteoblasts. These studies will elucidate novel regulatory mechanisms that control osteoblast function and bone formation in health and disease, and open new avenues in the pursuit of bone anabolics.
|
1 |
2009 |
Frenkel, Baruch |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Runx Proteins and Sex Steroid Signaling in Bone @ University of Southern California
Technical Abstract Sex Steroids and Runx Signaling in Bone The following contains proprietary/privileged information that Dr. Frenkel requests not to be released to persons outside the Government, except for purposes of review and evaluation. Runx2 is a master osteoblast transcription factor playing pivotal roles in skeletal development and homeostasis. In humans, Runx2 haplotypes contribute to variations in bone mass. Runx1, which is expressed in osteoblasts and shares similar DNA-binding properties with Runx2, has been implicated in bone metabolism as well. Sex steroid hormones and their receptors (SHRs) also play critical roles in bone health and disease, and are targets for existing and developing drugs that affect bone mass and fragility either positively or negatively. The proskeletal effects of sex steroids are mediated by anabolic effects in osteoblasts, but more importantly by attenuating bone resorption. The anti-resorptive effects of sex steroids are attributable to both direct pro-apoptotic action in osteoclasts and indirect inhibition of bone turnover via poorly understood mechanisms in osteoblasts and other mesenchymal cells. We found that the activated estrogen receptor [unreadable] (ER[unreadable]) and the androgen receptor (AR) each inhibits Runx2, and that AR, but not ER[unreadable], inhibits Runx1 as well. These inhibitory activities are important in light of recent data from [unreadable]1(I)collagen-Runx2 transgenic mice, indicating that Runx2 must be restrained in order to keep bone turnover in check and prevent osteoporosis. We therefore propose to investigate in depth the physical interactions between Runx proteins and SHRs, the mechanisms mediating the resulting inhibition of Runx2 and/or Runx1, and the in vivo physiological implications. This will be done by analyses of recombinant and transiently expressed proteins, as well as the endogenous SHR and Runx proteins in osteoblasts, including their associations with each other, with co- regulators, and with genomic Runx targets. Specific Aim 1 is to dissect the functional and molecular interactions between ER[unreadable] and Runx2. Specific Aim 2 is to dissect the functional and molecular interactions between AR and Runx2, as well as between AR and Runx1. Based on our preliminary data, we hypothesize the existence of both similar and unique features for each of these interactions. Specific Aim 3 is to test the ability of estrogens (and later androgens) to correct the hyper-osteoclastogenic phenotype of osteoblasts over- expressing Runx2 in vivo and when co-cultured with osteoclasts. Incorporated into Aims 1-3 are experiments addressing novel mechanisms of action of selective estrogen receptor modulators (SERMs). Like estradiol, SERMs promote a physical interaction between ER[unreadable] and Runx2. However, SERMs elicit different functional outcomes, possibly explaining the variable skeletal effects of these drugs. Our studies will provide novel insights into the regulation of skeletal metabolism by sex hormones, and will reveal commonalities and differences between the genders at the molecular level. They will decipher cryptic mechanisms of action of existing SERMs, and support the rationale development of novel ones, based on their influence on Runx proteins.
|
1 |
2010 — 2013 |
Frenkel, Baruch |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Sex Steroids and Runx Signaling in Bone @ University of Southern California
DESCRIPTION (provided by applicant): Runx2 is a master osteoblast transcription factor playing pivotal roles in skeletal development and homeostasis. In humans, Runx2 haplotypes contribute to variations in bone mass. Runx1, which is expressed in osteoblasts and shares similar DNA-binding properties with Runx2, has been implicated in bone metabolism as well. Sex steroid hormones and their receptors (SHRs) also play critical roles in bone health and disease, and are targets for drugs that affect bone mass and fragility either positively or negatively. The proskeletal effects of sex steroids are mediated by anabolic effects in osteoblasts, but more importantly by attenuating bone resorption. The anti-resorptive effects of sex steroids are attributable to both increasing osteoclast apoptosis and indirect inhibition of bone turnover via poorly understood mechanisms in osteoblasts and other mesenchymal cells. We found that the activated estrogen receptor a (ERa) and the androgen receptor (AR) each inhibits Runx2, and that AR, but not ERa, inhibits Runx1. These inhibitory activities are important in light of recent data from transgenic mice whose osteoblasts over-express either Runx2 or a dominant negative form of Runx2. Both mouse models indicate that restraining the activity of Runx2 helps keep bone turnover in check and prevent osteoporosis. We therefore propose to investigate in depth the physical interactions between Runx proteins and SHRs, the mechanisms mediating the resulting inhibition of Runx2 and/or Runx1, and the physiological implications. This will be done by analyses of recombinant and transiently expressed proteins, as well as the endogenous SHR and Runx proteins in osteoblasts, including their associations with each other, with co-regulators, and with genomic Runx targets. Specific Aim 1 is to dissect the functional and molecular interactions between ERa and Runx2. Specific Aim 2 is to dissect the functional and molecular interactions between AR and Runx2, as well as between AR and Runx1. Based on our preliminary data, we hypothesize the existence of both similar and unique features for each of these interactions. Specific Aim 3 is to establish in vivo the requirement for osteoblastic ERa signaling, and the timing during osteoblast differentiation, in which it confers protection on bone, and to test and characterize the anti-Runx2 anti-osteoclastogenic properties of osteoblastic SHR signaling in a co-culture setting. Incorporated into Aims 1-3 are experiments addressing novel mechanisms of action of selective estrogen receptor modulators (SERMs). Like estradiol, SERMs promote a physical interaction between ERa and Runx2. However, SERMs elicit different functional outcomes, possibly explaining the variable skeletal effects of these drugs. Our studies will provide novel insights into the regulation of skeletal metabolism by sex hormones, and will reveal commonalities and differences between the genders at the molecular level. They will decipher cryptic mechanisms of action of existing SERMs, and support the rationale development of novel ones, based on their influence on Runx proteins. PUBLIC HEALTH RELEVANCE: This project will unravel fundamental mechanisms of osteoporosis that occurs as sex hormones decline. It is based on the observation that Runx proteins, which control bone metabolism, interact with receptors for both estrogens and androgens. The proposed work can ultimately lead to the development of improved drugs to treat postmenopausal osteoporosis.
|
1 |
2017 |
Frenkel, Baruch Schones, Dustin Edward |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Modulation of Runx2 Activity by Era in Osteoblasts @ University of Southern California
ABSTRACT: Modulation of RUNX2 Activity by ER? in Osteoblasts The bone-sparing properties of estrogens are mediated by ER? in a variety of cell types, including cells of the osteoblast and osteoclast lineages, but the underlying molecular mechanisms are poorly understood. In particular, there are contrasting reports on how estradiol (E2) affects RUNX2, an osteoblast master regulator that has been implicated in human bone mass control through GWAS. RUNX2 plays critical roles in the osteoblast lineage to stimulate both autonomous cellular differentiation (and thus bone formation) and osteoblast-driven osteoclastogenesis (and thus bone resorption). While this project initiated with a focus on inhibition of RUNX2 by E2, the present renewal application aims to take genome-wide approaches to understand why E2 does not inhibit RUNX2-driven transcription uniformly. We show that RUNX2 activity upon various targets is modulated differently, with most targets inhibited, but others not inhibited and some even cooperatively stimulated, to various extents, by RUNX2 and E2. Locus-dependent differential modulation of RUNX2 is expected to ultimately change the balance between RUNX2-mediated osteoblast differentiation and RUNX2-mediated osteoblast-driven osteoclastogenesis. Our preliminary data also demonstrate that both raloxifen and lasofoxifene poorly mimic E2 in modulating activity of RUNX2 at different loci. Mechanisms underlying the locus- and ligand-dependent modulation of RUNX2 by ER? are completely unknown. Based on our preliminary results, we hypothesize that ER? differentially modulates RUNX2 across the osteoblast genome depending on local relative positions of sites occupied by RUNX2 and ER? and the ER ligand. Additionally, we hypothesize that local collaborating transcription factors (TFs), as well as specific cognate motif sequences for RUNX2, ER? and collaborating TFs shape local effects of ER? on RUNX2. We will first investigate by ChIP-seq analysis of histone marks how E2 modulates RUNX2-driven changes to the chromatin activation status at every genomic locus. The mutual effects of ER? and RUNX2 on occupying their target loci will also be determined genome-wide by ChIP-seq. Computational models will then be developed to explain the locus-specific combinatorial transcriptional regulation by RUNX2 and ER?. These models will be tested, first computationally and then experimentally, for their ability to predict effects of E2 on RUNX2 activity. Finally, since ER? in pre-osteoblasts protects female cortical bone in mice, and since raloxifen and lasofoxifene, SERMs commonly prescribed in the US and in Europe, respectively, only reduce the risk for vertebral (predominantly trabecular) osteoporotic fractures, but not non-vertebral fractures, we will also decode how raloxifene- and lasofoxifene-bound ER? modulate RUNX2 activity genome wide. Principal genomic determinants will be identified, which explain the differential modulation of RUNX2 activity as a function of the locus (different targets) and the ligand (E2 vs. raloxifen vs. lasofoxifene). This project will help understand how E2 differentially modulates RUNX2 activity at different loci to potentially alter the balance between bone formation and bone resorption. Gene sets will be identified where SERMs mimic, or do not mimic E2. While unveiling the underlying genomic codes, tools will be generated for future investigation of how E2, or any SERM, modulate RUNX2 activity at prototypic loci to differentially regulate specific gene sets.
|
1 |