RESUMEN
BACKGROUND: Hedgehog signaling is a stromal-mesenchymal pathway central to the development and homeostasis of both the prostate and the bone. Aberrant Hedgehog signaling activation has been associated with prostate cancer aggressiveness. We hypothesize that Hedgehog pathway is a candidate therapeutic target in advanced prostate cancer. We confirm increased Hedgehog signaling in advanced and bone metastatic castrate resistant prostate cancer and examine the pharmacodynamic effect of Smoothened inhibition by the novel reagent GDC-0449 in an experimental prostate cancer model. METHODS: Hedgehog signaling component expression was assessed in tissue microarrays of high grade locally advanced and bone metastatic disease. Male SCID mice subcutaneously injected with the bone forming xenograft MDA PCa 118b were treated with GDC-0449. Hedgehog signaling in the tumor microenvironment was assessed by proteomic and species specific RNA expression and compared between GDC-0449 treated and untreated animals. RESULTS: We observe Hedgehog signaling in high grade locally advanced and bone marrow infiltrating disease. Evidence of paracrine activation of Hedgehog signaling in the tumor xenograft, was provided by increased Sonic Hedgehog expression in human tumor epithelial cells, coupled with increased Gli1 and Patched1 expression in the murine stromal compartment, while normal murine stroma did not exhibit Hh signaling expression. GDC-0449 treatment attenuated Hh signaling as evidenced by reduced expression of Gli1 and Ptch1. Reduction in proliferation (Ki67) was observed with no change in tumor volume. CONCLUSIONS: GDC-0449 treatment is pharmacodynamically effective as evidenced by paracrine Hedgehog signaling inhibition and results in tumor cell proliferation reduction. Understanding these observations will inform the clinical development of therapy based on Hedgehog signaling inhibition.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Anilidas/farmacología , Antineoplásicos/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Osteogénesis/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Osificación Heterotópica/inducido químicamente , Osificación Heterotópica/metabolismo , Osificación Heterotópica/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Análisis de Matrices Tisulares , Trasplante Heterólogo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Previously the analysis of promoters regulated by endoplasmic reticulum (ER) stress identified a composite promoter element, ERSE that interacts with both CBF/NF-Y (CBF) and ATF6(N) transcription factors. This prompted us to investigate the underlying mechanism by which CBF, a ubiquitously binding transcription factor, specifically controls transcription activation during ER stress. The in vitro DNA binding study performed using purified recombinant proteins revealed that CBF specifically recruits ATF6(N) to ERSE DNA but it does not interact with ATF6(N) in absence of DNA binding. Inhibition of CBF binding resulted in a significant reduction of optimal transcription activation of cellular genes during ER stress. Analysis of cellular promoters by ChIP demonstrated that CBF is needed for recruitment of both ATF6(N) and TBP but not for either acetylation of histone H3-K9 or trimethylation of histone H3-K4 during ER stress. Together these study results reveal that CBF controls ER stress-inducible transcription through recruitment of both ATF6(N) and TBP but not through chromatin modifications. Our observations are in agreement with the results of recently published studies that have shown that CBF controls transcription of varieties of inducible promoters through recruitment of general transcription factors but not through acetylation of histone H4. These findings provide a paradigm of the function of CBF in inducible transcription.
Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Factor de Unión a CCAAT/metabolismo , Retículo Endoplásmico/patología , Proteína de Unión a TATA-Box/metabolismo , Transcripción Genética , Secuencia de Bases , Inmunoprecipitación de Cromatina , ADN/metabolismo , Chaperón BiP del Retículo Endoplásmico , Regulación Neoplásica de la Expresión Génica , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Activación Transcripcional/genéticaRESUMEN
Previous studies showed that binding of the CBF/NF-Y (CBF) transcription factor to cellular promoters is essential for cell proliferation. This observation prompted us to investigate the function of CBF in relation to cell cycle progression and in cell-cycle-regulated transcription. In this study, we used a tetracycline-inducible adenoviral vector to express a truncated CBF-B subunit, Bdbd, lacking a transcription activation domain in various mammalian cell lines. The Bdbd polypeptide interacts with cellular CBF-A/CBF-C and binds to promoters containing CBF-binding sites. Interestingly, expression of Bdbd in various mammalian cells resulted in the inhibition of cell proliferation and specific cell cycle arrest at G2/M phase. Gene expression analysis demonstrated that the expression of Bdbd strongly suppressed cell cycle-dependent transcription activation of Cyclin B1, Aurora A and CDK1 genes, key regulators for cell cycle progression at G2/M phase. Chromatin immunoprecipitation analysis showed that Bdbd significantly inhibited binding of TATA-binding protein, TBP to both Cyclin B1 and Aurora A promoters, but did not inhibit binding of E2F3 activator to Cyclin B1 promoter. This study suggested that the activation domain of CBF-B plays an essential role in the transcription activation of Cyclin B1 and Aurora A genes at G2/M phase, thus regulating cell cycle progression at G2/M phase.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , División Celular/genética , Ciclina B/genética , Fase G2/genética , Proteínas Serina-Treonina Quinasas/genética , Activación Transcripcional , Adenoviridae/genética , Aurora Quinasas , Factor de Unión a CCAAT/antagonistas & inhibidores , Factor de Unión a CCAAT/genética , Ciclina B1 , Vectores Genéticos , Células HeLa , Humanos , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Eliminación de Secuencia , Proteína de Unión a TATA-Box/metabolismo , Tetraciclina/farmacologíaRESUMEN
Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) is a validated treatment target for the treatment of metastatic castration-resistant prostate cancer (CRPC). Abiraterone acetate (AA) inhibits both 17α-hydroxylase (hydroxylase) and 17,20-lyase (lyase) reactions catalyzed by CYP17A1 and thus depletes androgen biosynthesis. However, coadministration of prednisone is required to suppress the mineralocorticoid excess and cortisol depletion that result from hydroxylase inhibition. VT-464, a nonsteroidal small molecule, selectively inhibits CYP17A1 lyase and therefore does not require prednisone supplementation. Administration of VT-464 in a metastatic CRPC patient presenting with high tumoral expression of both androgen receptor (AR) and CYP17A1, showed significant reduction in the level of both dehydroepiandrosterone (DHEA) and serum PSA. Treatment of a CRPC patient-derived xenograft, MDA-PCa-133 expressing H874Y AR mutant with VT-464, reduced the increase in tumor volume in castrate male mice more than twice as much as the vehicle (P < 0.05). Mass spectrometry analysis of post-treatment xenograft tumor tissues showed that VT-464 significantly decreased intratumoral androgens but not cortisol. VT-464 also reduced AR signaling more effectively than abiraterone in cultured PCa cells expressing T877A AR mutant. Collectively, this study suggests that VT-464 therapy can effectively treat CRPC and be used in precision medicine based on androgen receptor mutation status.
Asunto(s)
Naftalenos/administración & dosificación , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Triazoles/administración & dosificación , Acetato de Abiraterona/administración & dosificación , Andrógenos/biosíntesis , Animales , Biopsia , Línea Celular Tumoral , Deshidroepiandrosterona/química , Humanos , Hidrocortisona/sangre , Masculino , Espectrometría de Masas , Ratones , Ratones SCID , Trasplante de Neoplasias , Medicina de Precisión , Prednisona/administración & dosificación , Receptores Androgénicos/genética , Transducción de Señal , Esteroide 17-alfa-Hidroxilasa/metabolismoRESUMEN
PURPOSE: Morphologically heterogeneous prostate cancers that behave clinically like small-cell prostate cancers (SCPC) share their chemotherapy responsiveness. We asked whether these clinically defined, morphologically diverse, "aggressive variant prostate cancer (AVPC)" also share molecular features with SCPC. EXPERIMENTAL DESIGN: Fifty-nine prostate cancer samples from 40 clinical trial participants meeting AVPC criteria, and 8 patient-tumor derived xenografts (PDX) from 6 of them, were stained for markers aberrantly expressed in SCPC. DNA from 36 and 8 PDX was analyzed by Oncoscan for copy number gains (CNG) and losses (CNL). We used the AVPC PDX to expand observations and referenced publicly available datasets to arrive at a candidate molecular signature for the AVPC. RESULTS: Irrespective of morphology, Ki67 and Tp53 stained ≥10% cells in 80% and 41% of samples, respectively. RB1 stained <10% cells in 61% of samples and AR in 36%. MYC (surrogate for 8q) CNG and RB1 CNL showed in 54% of 44 samples each and PTEN CNL in 48%. All but 1 of 8 PDX bore Tp53 missense mutations. RB1 CNL was the strongest discriminator between unselected castration-resistant prostate cancer (CRPC) and the AVPC. Combined alterations in RB1, Tp53, and/or PTEN were more frequent in the AVPC than in unselected CRPC and in The Cancer Genome Atlas samples. CONCLUSIONS: Clinically defined AVPC share molecular features with SCPC and are characterized by combined alterations in RB1, Tp53, and/or PTEN.
Asunto(s)
Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Proteínas Supresoras de Tumor/genética , Biomarcadores de Tumor , Biopsia , Análisis por Conglomerados , Variaciones en el Número de Copia de ADN , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Mutación , Estadificación de Neoplasias , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/terapia , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismoRESUMEN
OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Guanyuan" (CV 4) and bilateral "Housanli" (ST 36) plus acupuncture stimulation of "Baihui" (GV 20) [an acupoint recipe for "Shuanggu Yitong" (strengthening both congenital foundation and the acquired constitution and regulating the yang-qi of the body] on the anti-fatigue ability and liver mitochondrial respiratory function in yang-deficiency ageing rats. METHODS: A total of 48 male SD rats were equally and randomly divided into normal control, model, EA and EA control groups. The aged yang-deficiency model was established by subcutaneous injection of D-galactose (125 mg/kg, once daily for 40 days) and sequential muscular injection of hydrocortisone (1.5 mg/100 g, once daily for 7 days). For rats of the normal control group, subcutaneous injection of same dose of normal saline was gi-ven. EA stimulation (2 Hz, 1 mA) of "Guanyuan" (CV 4) and bilateral "Housanli"(ST 36) and manual acupuncture stimulation of "Baihui" (GV 20) were given to rats of the EA group. For rats of the EA control group, EA of "Zhongji"(CV 3) and bilateral "Yinlinquan"(SP 9), and manual acupuncture stimulation of "Yintang" (EX-HN 3) were given. The treatment was conducted once daily, 6 time a week and continuously for 4 weeks. Rats of the normal control and model groups were grabbed and fixed in the same way. The anti-fatigue ability was evaluated by using the time of exhausted swimming task and liver mitochondrial respiratory function was detected by Clark oxygen electrode. RESULTS: In comparison with the normal control group, the exhausted swimming time was significantly shortened in the model group (P < 0.01). Compared with the model group, the exhausted swimming time was evidently prolonged in the EA group (P < 0.01) not in the EA control group (P > 0.05). As for the liver mitochondrial respi-ratory function, the oxygen consumption rate IV was obviously higher in the model group than in the normal control group (P < 0.01), and significantly lower in the EA group than in the model group (P < 0.01). Both liver mitochondrial respiratory control rate (RCR) and ratio of phosphorus/oxgen (P/O) were significantly lower in the model group than in the normal control group (P < 0.01), and considerably higher in the EA group than in the model group (P < 0.01, P < 0.05). Comparison between the EA and EA control groups showed that the effects of the former group were evidently superior to those of the latter group in prolonging the exhausted swimming time, lowering oxygen consumption rate IV and upregulating RCR and P/O (P < 0.05). CONCLUSION: Electroacupuncture of CV 4 and ST 36 plus manual acupuncture stimulation of GV 20 can improve the anti-fatigue capability in aging rats with yang-deficiency, which may be related to its effects in reducing liver mitochondrial oxygen consumption and increasing liver mitochondrial RCR and ratio of P/O.
Asunto(s)
Envejecimiento/metabolismo , Electroacupuntura , Fatiga/terapia , Hígado/metabolismo , Mitocondrias/metabolismo , Deficiencia Yang/terapia , Puntos de Acupuntura , Animales , Modelos Animales de Enfermedad , Fatiga/metabolismo , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Deficiencia Yang/metabolismoRESUMEN
PURPOSE: Small-cell prostate carcinoma (SCPC) morphology predicts for a distinct clinical behavior, resistance to androgen ablation, and frequent but short responses to chemotherapy. We sought to develop model systems that reflect human SCPC and can improve our understanding of its biology. EXPERIMENTAL DESIGN: We developed a set of castration-resistant prostate carcinomas xenografts and examined their fidelity to their human tumors of origin. We compared the expression and genomic profiles of SCPC and large-cell neuroendocrine carcinoma (LCNEC) xenografts to those of typical prostate adenocarcinoma xenografts. Results were validated immunohistochemically in a panel of 60 human tumors. RESULTS: The reported SCPC and LCNEC xenografts retain high fidelity to their human tumors of origin and are characterized by a marked upregulation of UBE2C and other mitotic genes in the absence of androgen receptor (AR), retinoblastoma (RB1), and cyclin D1 (CCND1) expression. We confirmed these findings in a panel of samples of CRPC patients. In addition, array comparative genomic hybridization of the xenografts showed that the SCPC/LCNEC tumors display more copy number variations than the adenocarcinoma counterparts. Amplification of the UBE2C locus and microdeletions of RB1 were present in a subset, but none displayed AR nor CCND1 deletions. The AR, RB1, and CCND1 promoters showed no CpG methylation in the SCPC xenografts. CONCLUSION: Modeling human prostate carcinoma with xenografts allows in-depth and detailed studies of its underlying biology. The detailed clinical annotation of the donor tumors enables associations of anticipated relevance to be made. Future studies in the xenografts will address the functional significance of the findings.
Asunto(s)
Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/metabolismo , Modelos Animales de Enfermedad , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Anciano , Animales , Carcinoma de Células Pequeñas/patología , Hibridación Genómica Comparativa , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Ratones , Persona de Mediana Edad , Neoplasias de la Próstata/patología , Análisis de Matrices Tisulares , Trasplante HeterólogoRESUMEN
PURPOSE: To study Wnt/ß-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the ß-catenin-androgen receptor (AR) interaction. EXPERIMENTAL DESIGN: We carried out ß-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of ß-catenin-mediated transcription), and sequenced the ß-catenin gene in MDA prostate cancer 118a, MDA prostate cancer 118b, MDA prostate cancer 2b, and PC-3 prostate cancer cells. We knocked down ß-catenin in AR-negative MDA prostate cancer 118b cells and carried out comparative gene-array analysis. We also immunohistochemically analyzed ß-catenin and AR in 27 bone metastases of human CRPCs. RESULTS: ß-Catenin nuclear accumulation and TOP-flash reporter activity were high in MDA prostate cancer 118b but not in MDA prostate cancer 2b or PC-3 cells. MDA prostate cancer 118a and MDA prostate cancer 118b cells carry a mutated ß-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA prostate cancer 118b cells with downregulated ß-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant ß-catenin. Finally, we found nuclear localization of ß-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher's exact test), suggesting that reduced AR expression enables Wnt/ß-catenin signaling. CONCLUSION: We identified a previously unknown downstream target of ß-catenin, HAS2, in prostate cancer, and found that high ß-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone metastatic prostate cancer. These findings may guide physicians in managing these patients.
Asunto(s)
Glucuronosiltransferasa/genética , Neoplasias de la Próstata/genética , Transducción de Señal/fisiología , beta Catenina/genética , Animales , Western Blotting , Neoplasias Óseas/secundario , Perfilación de la Expresión Génica , Glucuronosiltransferasa/metabolismo , Humanos , Hialuronano Sintasas , Inmunohistoquímica , Masculino , Ratones , Ratones SCID , Mutación , Neoplasias de la Próstata/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Androgénicos/biosíntesis , Receptores Androgénicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Heterólogo , beta Catenina/metabolismoRESUMEN
The enzyme 5α-reductase, which converts testosterone to dihydrotestosterone (DHT), performs key functions in the androgen receptor (AR) signaling pathway. The three isoenzymes of 5α-reductase identified to date are encoded by different genes: SRD5A1, SRD5A2, and SRD5A3. In this study, we investigated mechanisms underlying androgen regulation of 5α-reductase isoenzyme expression in human prostate cells. We found that androgen regulates the mRNA level of 5α-reductase isoenzymes in a cell type-specific manner, that such regulation occurs at the transcriptional level, and that AR is necessary for this regulation. In addition, our results suggest that AR is recruited to a negative androgen response element (nARE) on the promoter of SRD5A3 in vivo and directly binds to the nARE in vitro. The different expression levels of 5α-reductase isoenzymes may confer response or resistance to 5α-reductase inhibitors and thus may have importance in prostate cancer prevention.
Asunto(s)
Andrógenos/farmacología , Colestenona 5 alfa-Reductasa/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/prevención & control , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Animales , Línea Celular Tumoral , Colestenona 5 alfa-Reductasa/genética , Dactinomicina/farmacología , Dihidrotestosterona/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Especificidad de Órganos/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/genética , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Androgénicos/metabolismo , Elementos de Respuesta/genética , Transcripción Genética/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Induction of new bone formation is frequently seen in the bone lesions from prostate cancer. However, whether osteogenesis is necessary for prostate tumor growth in bone is unknown. Recently, 2 xenografts, MDA-PCa-118b and MDA-PCa-133, were generated from prostate cancer bone metastases. When implanted subcutaneously in severe combined immunodeficient (SCID) mice, MDA-PCa-118b induced strong ectopic bone formation while MDA-PCa-133 did not. To identify the factors that are involved in bone formation, we compared the expression of secreted factors (secretome) from MDA-PCa-118b and MDA-PCa-133 by cytokine array. We found that the osteogenic MDA-PCa-118b xenograft expressed higher levels of bone morphogenetic protein BMP4 and several cytokines including interleukin-8, growth-related protein (GRO), and CCL2. We showed that BMP4 secreted from MDA-PCa-118b contributed to about a third of the osteogenic differentiation seen in MDA-PCa-118b tumors. The conditioned media from MDA-PCa-118b induced a higher level of osteoblast differentiation, which was significantly reduced by treatment with BMP4 neutralizing antibody or the small molecule BMP receptor 1 inhibitor LDN-193189. BMP4 did not elicit an autocrine effect on MDA-PCa-118b, which expressed low to undetectable levels of BMP receptors. Treatment of SCID mice bearing MDA-PCa-118b tumors with LDN-193189 significantly reduced tumor growth. Thus, these studies support a role of BMP4-mediated osteogenesis in the progression of prostate cancer in bone.
Asunto(s)
Adenocarcinoma/secundario , Proteína Morfogenética Ósea 4/fisiología , Neoplasias Óseas/secundario , Proteínas de Neoplasias/fisiología , Osificación Heterotópica/etiología , Osteogénesis/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Animales , Comunicación Autocrina , Proteína Morfogenética Ósea 4/antagonistas & inhibidores , Proteína Morfogenética Ósea 4/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/antagonistas & inhibidores , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/biosíntesis , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/fisiopatología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/trasplante , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Citocinas/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Masculino , Ratones , Ratones SCID , Proteínas de Neoplasias/metabolismo , Osteoblastos/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Proteínas Recombinantes/farmacología , Tejido Subcutáneo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Our previous work has provided strong evidence that the transcription factor SOX9 is completely needed for chondrogenic differentiation and cartilage formation acting as a "master switch" in this differentiation. Heterozygous mutations in SOX9 cause campomelic dysplasia, a severe skeletal dysmorphology syndrome in humans characterized by a generalized hypoplasia of endochondral bones. To obtain insights into the logic used by SOX9 to control a network of target genes in chondrocytes, we performed a ChIP-on-chip experiment using SOX9 antibodies. METHODOLOGY/PRINCIPAL FINDINGS: The ChIP DNA was hybridized to a microarray, which covered 80 genes, many of which are involved in chondrocyte differentiation. Hybridization peaks were detected in a series of cartilage extracellular matrix (ECM) genes including Col2a1, Col11a2, Aggrecan and Cdrap as well as in genes for specific transcription factors and signaling molecules. Our results also showed SOX9 interaction sites in genes that code for proteins that enhance the transcriptional activity of SOX9. Interestingly, a strong SOX9 signal was also observed in genes such as Col1a1 and Osx, whose expression is strongly down regulated in chondrocytes but is high in osteoblasts. In the Col2a1 gene, in addition to an interaction site on a previously identified enhancer in intron 1, another strong interaction site was seen in intron 6. This site is free of nucleosomes specifically in chondrocytes suggesting an important role of this site on Col2a1 transcription regulation by SOX9. CONCLUSIONS/SIGNIFICANCE: Our results provide a broad understanding of the strategies used by a "master" transcription factor of differentiation in control of the genetic program of chondrocytes.