Wound Healing-related Functions of the p160 Steroid Receptor Coactivator Family.

Multicellular organisms have evolved sophisticated mechanisms to recover and maintain original tissue functions following injury. Injury responses require a robust transcriptomic response associated with cellular reprogramming involving complex gene expression programs critical for effective tissue repair following injury. Steroid receptor coactivators (SRCs) are master transcriptional regulators of cell-cell signaling that is integral for embryogenesis, reproduction, normal physiological function, and tissue repair following injury. Effective therapeutic approaches for facilitating improved tissue regeneration and repair will likely involve temporal and combinatorial manipulation of cell-intrinsic and cell-extrinsic factors. Pleiotropic actions of SRCs that are critical for wound healing range from immune regulation and angiogenesis to maintenance of metabolic regulation in diverse organ systems. Recent evidence derived from studies of model organisms during different developmental stages indicates the importance of the interplay of immune cells and stromal cells to wound healing. With SRCs being the master regulators of cell-cell signaling integral to physiologic changes necessary for wound repair, it is becoming clear that therapeutic targeting of SRCs provides a unique opportunity for drug development in wound healing. This review will provide an overview of wound healing-related functions of SRCs with a special focus on cellular and molecular interactions important for limiting tissue damage after injury. Finally, we review recent findings showing stimulation of SRCs following cardiac injury with the SRC small molecule stimulator MCB-613 can promote cardiac protection and inhibit pathologic remodeling after myocardial infarction.

Transient activation of AMPK preceding left ventricular pressure overload reduces adverse remodeling and preserves left ventricular function.

Coordinated changes in signaling pathways and gene expression in hearts subjected to prolonged stress maintain cardiac function. Loss of steroid receptor coactivator-2 (SRC-2) results in a reversal to the fetal gene program and disrupts the response to pressure overload, accompanied by prominent effects on metabolism and growth signaling, including increased AMPK activation. We proposed that early metabolic stress driven by AMPK activation induces contractile dysfunction in mice lacking SRC-2. We used 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK transiently before transverse aortic constriction (TAC) in wild-type and cardiomyocyte-specific SRC-2 knockout (CKO) animals. In contrast to AMPK activities during stress, in unstressed hearts, AICAR induced a mild activation of Akt signaling, and, in SRC-2-CKO mice, partially relieved an NAD+ deficiency and increased antioxidant signaling. These molecular changes translated to a mild hypertrophic response to TAC with decreased maladaptive remodeling, including markedly decreased fibrosis. Additionally, preactivation of AMPK in SRC-2-CKO mice was accompanied by a dramatic improvement in cardiac function compared with saline-treated SRC-2-CKO mice. Our results show that altered molecular signaling before stress onset has extended effects on sustained cardiac stress responses, and prestress modulation of transient growth and metabolism pathways may control those effects.-Nam, D. H., Kim, E., Benham, A., Park, H.-K., Soibam, B., Taffet, G. E., Kaelber, J. T., Suh, J. H., Taegtmeyer, H., Entman, M. L., Reineke, E. L. Transient activation of AMPK preceding left ventricular pressure overload reduces adverse remodeling and preserves left ventricular function.

Retinoid signaling controlled by SRC-2 in decidualization revealed by transcriptomics

Establishment of a successful pregnancy requires not only implantation of a healthy embryo into a receptive uterus but also progesterone receptor (PGR)-dependent transformation of endometrial stromal cells (ESCs) into specialized decidual cells. Decidual cells support the developing embryo and are critical for placentation. We have previously shown that a known transcriptional coregulator of the PGR, steroid receptor coactivator-2 (SRC-2), is a critical driver of endometrial decidualization in both human and mouse endometrium. However, the full spectrum of genes transcriptionally controlled by SRC-2 in decidualizing ESCs has not been identified. Therefore, using an RNA- and chromatin immunoprecipitation-sequencing approach, we have identified the transcriptome of decidualizing human ESCs (hESCs) that requires SRC-2. We revealed that the majority of hESC genes regulated by SRC-2 are associated with decidualization. Over 50% of SRC-2-regulated genes are also controlled by the PGR. While ontology analysis showed that SRC-2-dependent genes are functionally linked to signaling processes known to underpin hESC decidualization, cell membrane processes were significantly enriched in this analysis. Follow-up studies showed that retinoid signaling is dependent on SRC-2 during hESC decidualization. Specifically, SRC-2 is required for full induction of the retinol transporter, stimulated by retinoic acid 6 (STRA6), which is essential for hESC decidualization. Together our findings show that a critical subset of genes transcriptionally reprogramed by PGR during hESC decidualization requires SRC-2. Among the multiple genes, pathways and networks that are dependent on SRC-2 during hESC decidualization, first-line analysis supports a critical role for this coregulator in maintaining retinoid signaling during progesterone-driven decidualization.

Genetic and Environmental Models of Circadian Disruption Link SRC-2 Function to Hepatic Pathology.

Circadian rhythmicity is a fundamental process that synchronizes behavioral cues with metabolic homeostasis. Disruption of daily cycles due to jet lag or shift work results in severe physiological consequences including advanced aging, metabolic syndrome, and even cancer. Our understanding of the molecular clock, which is regulated by intricate positive feedforward and negative feedback loops, has expanded to include an important metabolic transcriptional coregulator, Steroid Receptor Coactivator-2 (SRC-2), that regulates both the central clock of the suprachiasmatic nucleus (SCN) and peripheral clocks including the liver. We hypothesized that an environmental uncoupling of the light-dark phases, termed chronic circadian disruption (CCD), would lead to pathology similar to the genetic circadian disruption observed with loss of SRC-2 We found that CCD and ablation of SRC-2 in mice led to a common comorbidity of metabolic syndrome also found in humans with circadian disruption, non-alcoholic fatty liver disease (NAFLD). The combination of SRC-2(-/-) and CCD results in a more robust phenotype that correlates with human non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) gene signatures. Either CCD or SRC-2 ablation produces an advanced aging phenotype leading to increased mortality consistent with other circadian mutant mouse models. Collectively, our studies demonstrate that SRC-2 provides an essential link between the behavioral activities influenced by light cues and the metabolic homeostasis maintained by the liver.

Fetal-to-maternal signaling to initiate parturition.

Multiple processes are capable of activating the onset of parturition; however, the specific contributions of the mother and the fetus to this process are not fully understood. In this issue of the JCI, Gao and colleagues present evidence that steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2) regulate surfactant protein-A (SP-A) and platelet-activating factor (PAF) expression, which increases in the developing fetal lung. WT dams crossed with males deficient for both SRC-1 and SRC-2 had suppressed myometrial inflammation, increased serum progesterone, and delayed parturition, which could be reconciled by injection of either SP-A or PAF into the amnion. Together, the results of this study demonstrate that the fetal lungs produce signals to initiate labor in the mouse. This work underscores the importance of the fetus as a contributor to the onset of murine, and potentially human, parturition.

Steroid receptor coactivators 1 and 2 mediate fetal-to-maternal signaling that initiates parturition.

The precise mechanisms that lead to parturition are incompletely defined. Surfactant protein-A (SP-A), which is secreted by fetal lungs into amniotic fluid (AF) near term, likely provides a signal for parturition; however, SP-A-deficient mice have only a relatively modest delay (~12 hours) in parturition, suggesting additional factors. Here, we evaluated the contribution of steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2), which upregulate SP-A transcription, to the parturition process. As mice lacking both SRC-1 and SRC-2 die at birth due to respiratory distress, we crossed double-heterozygous males and females. Parturition was severely delayed (~38 hours) in heterozygous dams harboring SRC-1/-2-deficient embryos. These mothers exhibited decreased myometrial NF-κB activation, PGF2α, and expression of contraction-associated genes; impaired luteolysis; and elevated circulating progesterone. These manifestations also occurred in WT females bearing SRC-1/-2 double-deficient embryos, indicating that a fetal-specific defect delayed labor. SP-A, as well as the enzyme lysophosphatidylcholine acyltransferase-1 (LPCAT1), required for synthesis of surfactant dipalmitoylphosphatidylcholine, and the proinflammatory glycerophospholipid platelet-activating factor (PAF) were markedly reduced in SRC-1/-2-deficient fetal lungs near term. Injection of PAF or SP-A into AF at 17.5 days post coitum enhanced uterine NF-κB activation and contractile gene expression, promoted luteolysis, and rescued delayed parturition in SRC-1/-2-deficient embryo-bearing dams. These findings reveal that fetal lungs produce signals to initiate labor when mature and that SRC-1/-2-dependent production of SP-A and PAF is crucial for this process.

Coactivator SRC-2-dependent metabolic reprogramming mediates prostate cancer survival and metastasis.

Metabolic pathway reprogramming is a hallmark of cancer cell growth and survival and supports the anabolic and energetic demands of these rapidly dividing cells. The underlying regulators of the tumor metabolic program are not completely understood; however, these factors have potential as cancer therapy targets. Here, we determined that upregulation of the oncogenic transcriptional coregulator steroid receptor coactivator 2 (SRC-2), also known as NCOA2, drives glutamine-dependent de novo lipogenesis, which supports tumor cell survival and eventual metastasis. SRC-2 was highly elevated in a variety of tumors, especially in prostate cancer, in which SRC-2 was amplified and overexpressed in 37% of the metastatic tumors evaluated. In prostate cancer cells, SRC-2 stimulated reductive carboxylation of α-ketoglutarate to generate citrate via retrograde TCA cycling, promoting lipogenesis and reprogramming of glutamine metabolism. Glutamine-mediated nutrient signaling activated SRC-2 via mTORC1-dependent phosphorylation, which then triggered downstream transcriptional responses by coactivating SREBP-1, which subsequently enhanced lipogenic enzyme expression. Metabolic profiling of human prostate tumors identified a massive increase in the SRC-2-driven metabolic signature in metastatic tumors compared with that seen in localized tumors, further implicating SRC-2 as a prominent metabolic coordinator of cancer metastasis. Moreover, SRC-2 inhibition in murine models severely attenuated the survival, growth, and metastasis of prostate cancer. Together, these results suggest that the SRC-2 pathway has potential as a therapeutic target for prostate cancer.

NCOA3 is a selective co-activator of estrogen receptor α-mediated transactivation of PLAC1 in MCF-7 breast cancer cells.

BACKGROUND: The placenta-specific 1 (PLAC1) gene encodes a membrane-associated protein which is selectively expressed in the placental syncytiotrophoblast and in murine fetal tissues during embryonic development. In contrast to its transcriptional repression in all other adult normal tissues, PLAC1 is frequently activated and highly expressed in a variety of human cancers, in particular breast cancer, where it associates with estrogen receptor α (ERα) positivity. In a previous study, we showed that ERα-signaling in breast cancer cells transactivates PLAC1 expression in a non-classical pathway. As the members of the p160/nuclear receptor co-activator (NCOA) family, NCOA1, NCOA2 and NCOA3 are known to be overexpressed in breast cancer and essentially involved in estrogen-mediated cancer cell proliferation we asked if these proteins are involved in the ERα-mediated transactivation of PLAC1 in breast cancer cells. METHODS: Applying quantitative real-time RT-PCR (qRT-PCR), Western Blot analysis and chromatin immunoprecipitation, we analyzed the involvement of NCOA1, NCOA2, NCOA3 in the ERα-mediated transactivation of PLAC1 in the breast cancer cell lines MCF-7 and SK-BR-3. RNAi-mediated silencing of NCOA3, qRT-PCR, Western blot analysis and ERα activation assays were used to examine the role of NCOA3 in the ERα-mediated regulation of PLAC1 in further detail. Transcript expression of NCOA3 and PLAC1 in 48 human breast cancer samples was examined by qRT-PCR and statistical analysis was performed using Student's t-test. RESULTS: We detected selective recruitment of NCOA3 but not NCOA1 or NCOA2 to the PLAC1 promoter only in ERα-positive MCF-7 cells but not in ERα-negative SK-BR-3 breast cancer cells. In addition, we demonstrate that silencing of NCOA3 results in a remarkable decrease of PLAC1 expression levels in MCF-7 cells which cannot be restored by treatment with estradiol (E2). Moreover, significant higher transcript levels of PLAC1 were found only in ERα-positive human breast cancer samples which also show a NCOA3 overexpression. CONCLUSIONS: In this study, we identified NCOA3 as a selective co-activator of ERα-mediated transactivation of PLAC1 in MCF-7 breast cancer cells. Our data introduce PLAC1 as novel target gene of NCOA3 in breast cancer, supporting the important role of both factors in breast cancer biology.

The transcription intermediary factor 1ß coactivates the androgen receptor.

The androgen receptor (AR) is a ligand-inducible transcription factor. Its transcription activation domain consists of the two transcription activation units called Tau-1 and Tau- 5. Tau-5 interacts with p160 coactivators like the transcription intermediary factor 2 (TIF2), which in their turn recruit histone modifiers and chromatin-remodelling complexes. The mechanism of action of Tau-1, however, remains elusive. Here, we demonstrate that transcription intermediary factor 1ß (TIF1ß) can induce the activity of the AR up to five fold when tested in vitro. Although there is no evidence for direct interactions between TIF1ß and AR, mutation studies show that the activity of TIF1ß depends on the integrity of Tau-1 in AR on the one hand, and the so-called tripartite motif domain in TIF1ß on the other. Surprisingly, the coactivation by TIF1ß via Tau-1 seems additive rather than cooperative with the AR coactivation by TIF2. Some mutations naturally occurring in androgen-insensitivity syndrome patients that reside in Tau-1 seem to impair the TIF1ß coactivation of the AR, indicating that TIF1ß could also be relevant for the in vivo androgen response in humans. Moreover, since TIF1ß is well expressed in prostate cancer cells, its functional interaction with androgen signalling could in the long run be a therapeutic target for this disease.

Steroid receptor coactivator 2 modulates steroid-dependent male sexual behavior and neuroplasticity in Japanese quail (Coturnix japonica).

Steroid receptor coactivators are necessary for efficient transcriptional regulation by ligand-bound nuclear receptors, including estrogen and androgen receptors. Steroid receptor coactivator-2 (SRC-2) modulates estrogen- and progesterone-dependent sexual behavior in female rats but its implication in the control of male sexual behavior has not been studied to our knowledge. We cloned and sequenced the complete quail SRC-2 transcript and showed by semi-quantitative PCR that SRC-2 expression is nearly ubiquitous, with high levels of expression in the kidney, cerebellum and diencephalon. Real-time quantitative PCR did not reveal any differences between intact males and females the medial preoptic nucleus (POM), optic lobes and cerebellum. We next investigated the physiological and behavioral role of this coactivator using in vivo antisense oligonucleotide techniques. Daily injections in the third ventricle at the level of the POM of locked nucleic acid antisense targeting SRC-2 significantly reduced the expression of testosterone-dependent male-typical copulatory behavior but no inhibition of one aspect of the appetitive sexual behavior was observed. The volume of POM, defined by aromatase-immunoreactive cells, was markedly decreased in animals treated with antisense as compared with controls. These results demonstrate that SRC-2 plays a prominent role in the control of steroid-dependent male sexual behavior and its associated neuroplasticity in Japanese quail.

Glucocorticoid receptor phosphorylation modulates transcription efficacy through GRIP-1 recruitment.

The role of GR phosphorylation in modulating GR-mediated transcription is not fully understood. Here we show that the hGR is rapidly phosphorylated at S211 and S226 in response to the synthetic agonist dexamethasone (dex) in COS-1 cells. Using a triple phosphorylation mutant hGR construct, we demonstrate that phosphorylation at one or more S residues (from S203, S211, and S226) is required for maximal hGR-mediated transcriptional activation on the MMTV promoter in response to dex in COS-1 cells, but that this effect is promoter selective. Phosphorylation at these residues does not affect unliganded or agonist-induced hGR degradation, suggesting that the mechanism whereby hGR phosphorylation at these residues regulates GR-mediated transactivation via a GRE does not involve changes in GR half-life. We have previously shown a direct correlation between efficacy for transactivation and interaction of the hGR with glucocorticoid receptor interacting protein-1 (GRIP-1). Here we show by pull-down assays in the absence and presence of glucocorticoid response elements (GREs) that phosphorylation of the hGR is required for GR-GRIP-1 interaction. Chromatin immunoprecipitation (ChIP) assays revealed that hGR phosphorylation at one or more S residues (from S226, S211, and S203) is required for the recruitment of GRIP-1 to the synthetic MMTV promoter as well as to the endogenous GRE-containing glucocorticoid-induced leucine zipper (GILZ) promoter in intact COS-1 cells, but not for nuclear localization. Our results support the conclusion that phosphorylation at S203, S211, and/or S226 of the hGR is required for a maximal transcriptional response via the synthetic MMTV and endogenous GILZ GREs in COS-1 cells, to enable recruitment of GRIP-1 to the hGR.

Normal and cancer-related functions of the p160 steroid receptor co-activator (SRC) family.

The three homologous members of the p160 SRC family (SRC1, SRC2 and SRC3) mediate the transcriptional functions of nuclear receptors and other transcription factors, and are the most studied of all the transcriptional co-activators. Recent work has indicated that the SRCgenes are subject to amplification and overexpression in various human cancers. Some of the molecular mechanisms responsible for SRC overexpression, along with the mechanisms by which SRCs promote breast and prostate cancer cell proliferation and survival, have been identified, as have the specific contributions of individual SRC family members to spontaneous breast and prostate carcinogenesis in genetically manipulated mouse models. These studies have identified new challenges for cancer research and therapy.

Skeletal consequences of deletion of steroid receptor coactivator-2/transcription intermediary factor-2.

Both estrogen receptor (ER) and peroxisome proliferator-activated receptor gamma (PPARgamma) regulate bone metabolism, and because steroid receptor coactivator (SRC)-2 (TIF-2) enhances ER and PPARgamma activity, we examined the consequences of deletion of SRC-2 on bone using SRC-2 knock out (KO) mice. Loss of SRC-2 resulted in increased bone mass, with SRC-2 KO mice having 80% higher trabecular bone volume as compared with wild type mice. SRC-2 KO mice also had a marked decrease (by 50%) in bone marrow adipocytes. These data suggested that marrow precursor cells in the SRC-2 KO mice may be resistant to the inhibitory effects of endogenous PPARgamma ligands on bone formation. Consistent with this, compared with cultures from wild type mice, marrow stromal cultures from SRC-2 KO mice formed significantly more mineralized nodules (by 3-fold) in the presence of the PPARgamma agonist, rosiglitazone. Using chromatin immunoprecipitation analysis, we demonstrated that in bone marrow stromal cells, loss of SRC-2 leads to destabilization of the transcription complex at the peroxisome proliferator response elements of a number of PPARgamma target genes, resulting in an overall decrease in the expression of adipocyte-related genes and a marked decrease in adipocyte development. Using ovariectomy with or without estrogen replacement, we also demonstrated that SRC-2 KO mice were partially resistant to the skeletal actions of estrogen. Collectively, these findings indicate that loss of SRC-2 leads to partial skeletal resistance to the ER and PPARgamma, but resistance to PPARgamma is dominant, leading to increased bone mass. Modulating SRC-2 action may, thus, represent a novel therapeutic target for osteoporosis.

Interleukin-6 increases prostate cancer cells resistance to bicalutamide via TIF2.

The standard treatment for advanced, androgen-responsive prostate cancer is androgen deprivation therapy with or without a nonsteroidal antiandrogen, such as bicalutamide. Although maximal androgen blockade exhibits favorable responses in the majority of patients, prostate cancer eventually progresses to an androgen-refractory stage. The mechanism underlying bicalutamide resistance in the course of prostate cancer progression is incompletely understood. However, interleukin-6 (IL-6) plays a critical role in the development and progression of CRPC. Herein, we explored an association between IL-6 and bicalutamide resistance. To study this, series of lower and higher passages of LNCaP cell sublines generated by long-term exposure to IL-6 were used. The cells from higher passages of LNCaP treated with IL-6 developed resistance to bicalutamide treatment compared with parental LNCaP cells. The levels of transcriptional intermediary factor 2 (TIF2) in IL-6-treated LNCaP cells were found to be significantly higher than parental LNCaP cells. Down-regulation of TIF2 expression via short hairpin RNA in IL-6-treated LNCaP cells sensitized these cells to bicalutamide treatment, whereas overexpression of TIF2 in the parental LNCaP cells increased resistance to bicalutamide. Furthermore, overexpression of IL-6 attenuated bicalutamide-mediated blockage of androgen-induced androgen receptor nuclear translocation and recruitment. These results show that overexpression of IL-6 increases the resistance of prostate cancer cells to bicalutamide via TIF2. Overexpression of IL-6 not only plays an important role in prostate cancer progression but also contributes to bicalutamide resistance. Our studies suggest that bicalutamide-IL-6-targeted adjunctive therapy may lead to a more effective intervention than bicalutamide alone.

Unique roles of p160 coactivators for regulation of breast cancer cell proliferation and estrogen receptor-alpha transcriptional activity.

Each of the three members of the p160 steroid receptor coactivator (SRC) family of coactivators (SRC-1, SRC-2 and SRC-3) stimulates estrogen receptor (ER)-alpha function in trans-activation assays. Consequently, we sought to elucidate their contributions to the ER-regulated processes of cell proliferation, apoptosis, and the expression of ERalpha target genes in MCF-7 breast cancer cells. The small interfering RNA depletion of SRC-2 or SRC-3 but not SRC-1 inhibited growth of MCF-7 cells, and this was reflected in decreased cell cycle progression and increased apoptosis in SRC-2- or SRC-3-depleted cells as well as a reduction in ERalpha transcriptional activity measured on a synthetic reporter gene. However, only SRC-3 depletion blocked estradiol stimulated cell proliferation. Depletion of SRC-1 did not affect these events, and together this reveals functional differences between each of the three SRC family coactivators. Regulation of the endogenous ERalpha target gene, c-myc was not affected by depletion of any of the p160 coactivators although depletion of each of them decreased pS2 mRNA expression in estradiol-treated MCF-7 cells. Moreover, progesterone receptor and cyclin D1 gene expression were decreased in SRC-3 small interfering RNA-treated cells. Expression of mRNA and protein levels for the antiapoptotic gene, Bcl-2 was dependent on SRC-3 expression, whereas Bcl-2 protein but not mRNA expression also was sensitive to SRC-1 depletion. Together these data indicate that the closely related p160 coactivators are not functionally redundant in breast cancer cells because they play gene-specific roles in regulating mRNA and protein expression, and they therefore are likely to make unique contributions to breast tumorigenesis.

A role for the transcription intermediary factor 2 in zebrafish myelopoiesis.

OBJECTIVE: TIF2 is fused with MOZ in the inv(8)(p11q13) acute myeloid leukemia. TIF2, member of the p160 family, is a histone acetyl transferase (HAT). Deletion of p160 genes were performed in mice. Some observations suggest that p160 family members may perform overlapping functions in mice. Therefore, we decided to choose the zebrafish model to study TIF2. The aim of this study was to characterize the role of this HAT during embryonic development. MATERIAL AND METHODS: We use antisense, morpholino-modified oligomers to transiently knockdown tif2 gene, thus determining whether TIF2 plays a role in zebrafish early development. RESULTS: We show that tif2 is involved in embryogenesis and in primitive hematopoiesis. tif2-knockdown zebrafish embryos are smaller than controls, they demonstrate shorter tails, they display notochord deformation and they exhibit U-shaped tail somites. A synthetic RNA encoding human TIF2 rescues the tif2-knockdown phenotype. Analysis of fli1 expression by whole-mount in situ hybridization indicates normal angioblast specification, but altered localization of intersomitic vessels. The posterior intermediate cell mass, in which a part of primitive hematopoiesis occurs, is altered in tif2 morphants and whole-mount in situ hybridization analyses of l-plastin and mpx expression suggest a specific inhibition of granulocytic and macrophagic differentiation at late stages. CONCLUSION: These data indicate an important role for TIF2 in zebrafish primitive myelopoiesis.

Regulation of vascular endothelial growth factor D by orphan receptors hepatocyte nuclear factor-4 alpha and chicken ovalbumin upstream promoter transcription factors 1 and 2.

Vascular endothelial growth factor D has recently been linked to the control of lymphangiogenesis and lymphatic metastasis. The molecular determinants regulating vegf-D gene transcription, however, have not yet been identified. After isolation of 2 kb of 5'-flanking DNA of the human vegf-D gene, we identified a novel, atypical direct repeat (DR) element consisting of a consensus half-site (AGGTCA) at -125/-119 and a degenerated DR half-site (ATGTTA) at -99/-94 as sufficient and necessary for vegf-D transcription. The vegf-D DR element is bound and activated by the orphan receptors hepatocyte nuclear factor 4 alpha (HNF-4 alpha) and chicken ovalbumin upstream promoter transcription factor (COUP-TF)-1/COUP-TF2. Additionally, chromatin immunoprecipitation assays identified transcriptional coactivators cyclic AMP-responsive element binding protein-binding protein and glucocorticoid receptor interacting protein 1 at the vegf-D DR element and functional assays confirmed their stimulatory effect on the vegf-D promoter. Histone deacetylase inhibition by trichostatin A led to accumulation of acetylated histones H3/H4 at the vegf-D promoter, up-regulation of vegf-D mRNA levels, and transactivation of vegf-D promoter reporter gene constructs in cancer cell lines. This study for the first time describes the molecular determinants in cis and trans controlling vegf-D gene transcription and identifies interaction of HNF-4 alpha and COUP-TF1/COUP-TF2 with a proximal, atypical DR element as indispensable for vegf-D transcription. Moreover, our findings suggest that epigenetic control of histone acetylation represents an important determinant of vegf-D gene expression in cancer cells. These results provide novel insights into the molecular machinery controlling vegf-D gene expression and may add to a better understanding of the regulation of lymphangiogenesis in vascular development and cancer.

The p160 steroid receptor coactivator 2, SRC-2, regulates murine endometrial function and regulates progesterone-independent and -dependent gene expression.

The role of the p160 steroid receptor coactivator 2 (SRC-2) in the regulation of uterine function and progesterone (P4) signaling was investigated by determining the expression pattern of SRC-2 in the murine uterus during pregnancy and the impact of SRC-2 ablation on uterine function and global uterine gene expression in response to progesterone. SRC-2 is expressed in the endometrial luminal and glandular epithelium from pregnancy d 0.5. SRC-2 is then expressed in the endometrial stroma on pregnancy d 2.5-3.5. Once the embryo is implanted, SRC-2 is expressed in the endometrial stromal cells in the secondary decidual zone. This compartmental expression of SRC-2 can be mimicked by treatment of ovariectomized mice with estrogen and P4. Ablation of SRC-2 in the uterus resulted in a significant reduction in the ability of the uterus to undergo a hormonally induced decidual reaction. Microarray analysis of RNA from uteri of wild-type and SRC-2(-/-) mice treated with vehicle or P4 showed that SRC-2 was involved in the ability of progesterone to repress specific genes. This microarray analysis also revealed that the uteri of SRC-2(-/-) mice showed alterations in genes involved in estrogen receptor, Wnt, and bone morphogenetic protein signaling. This analysis indicates that SRC-2 regulates uterine function by modulating the regulation of developmentally important signaling molecules and the ability of P4 to repress specific genes.

Progesterone-action in the murine uterus and mammary gland requires steroid receptor coactivator 2: relevance to the human.

The importance of the progesterone receptor (PR) in female reproductive and mammary gland biology is well recognized; however, the coregulators selectively enlisted by PR have yet to be comprehensively defined in vivo. To evaluate the involvement of steroid receptor coactivator (SRC)/p160 family members in these physiological systems, a mouse model (PRCre/+SRC-2flox/flox) was generated in which SRC-2 function was ablated specifically in cell-types that express the PR. Although PRCre/+SRC-2flox/flox ovarian activity was normal, uterine function was severely compromised. Absence of SRC-2 in PR positive uterine cells led to an early block in embryo implantation, a defect not ascribed to SRC-1 or -3 knockouts. While the PRCre/+SRC-2flox/flox uterus can display a partial decidual response, removal of SRC-1 in the PRCre/+SRC-2flox/flox uterus results in a block in decidualization, confirming that uterine SRC-2 and -1 are both necessary for PR-mediated transcriptional responses which lead to complete decidualization. The absence of significant branching and alveolar morphogenesis in the hormone-treated PRCre/+SRC-2flox/flox mammary gland establishes an important role for mammary SRC-2 in cellular proliferative programs that require PR. Finally, the observation that SRC-2 is also expressed in many of the same cell-types in the human, underscores the importance of further study of this coregulator's role in both peri-implantation biology and mammary development.