Klf10 inhibits IL-12p40 production in macrophage colony-stimulating factor-induced mouse bone marrow-derived macrophages.

Bone marrow-derived macrophages (BMMs) treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), differentiate into GM-CSF-induced mouse bone marrow-derived macrophages (GM-BMMs) or M-CSF-induced mouse bone marrow-derived macrophages (M-BMMs), which have an M1 or M2 profile, respectively. GM-BMMs produce large amounts of proinflammatory cytokines and mediate resistance to pathogens, whereas M-BMMs produce antiinflammatory cytokines that contribute to tissue repair and remodeling. M-BMMs stimulated with lipopolysaccharide (LPS) are in an antiinflammatory state, with an IL-12(low) IL-10(high) phenotype. However, the regulation of this process remains unclear. Klf10 belongs to the family of Krüppel-like transcription factors and was initially described as a TGF-ß inducible early gene 1. IL-12p40 is upregulated in LPS-stimulated M-BMMs from Klf10-deficient mice, but downregulated during Klf10 overexpression. Klf11, another member of the Krüppel-like factor family, can also repress the production of IL-12p40. Furthermore, Klf10 binds to the CACCC element of the IL-12p40 promoter and inhibits its transcription. We have therefore identified Klf10 as a transcription factor that regulates the expression of IL-12p40 in M-BMMs.

ERß1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer.

BACKGROUND: The role and clinical value of ERß1 expression is controversial and recent data demonstrates that many ERß antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERß1 expression across all sub-types of breast cancer using a validated antibody and determine the roles of this receptor in mediating response to multiple forms of endocrine therapy both in the presence and absence of ERα expression. METHODS: Nuclear and cytoplasmic expression patterns of ERß1 were analyzed in three patient cohorts, including a retrospective analysis of a prospective adjuvant tamoxifen study and a triple negative breast cancer cohort. To investigate the utility of therapeutically targeting ERß1, we generated multiple ERß1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERß-specific agonist exposure. RESULTS: Nuclear ERß1 was shown to be expressed across all major sub-types of breast cancer, including 25% of triple negative breast cancers and 33% of ER-positive tumors, and was associated with significantly improved outcomes in ERα-positive tamoxifen-treated patients. In agreement with these observations, ERß1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs). However, in the absence of ERα expression, ERß-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective. CONCLUSIONS: Using a validated antibody, we have confirmed that nuclear ERß1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERß appears to be a novel therapeutic target. However, the efficacy of SERMs and ERß-specific agonists differ as a function of ERα expression.

TGF-ß1 signaling and Krüppel-like factor 10 regulate bone marrow-derived proangiogenic cell differentiation, function, and neovascularization.

Emerging evidence demonstrates that proangiogenic cells (PACs) originate from the BM and are capable of being recruited to sites of ischemic injury where they contribute to neovascularization. We previously determined that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitor cells (GMPs) differentiate into PACs and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF-ß1-responsive Krüppel- like factor, KLF10, is strongly expressed in PACs derived from CMPs and GMPs, ∼ 60-fold higher than in progenitors lacking PAC markers. KLF10(-/-) mice present with marked defects in PAC differentiation, function, TGF-ß responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10(-/-) PACs. Overexpression studies revealed that KLF10 could rescue PAC formation from TGF-ß1(+/-) CMPs and GMPs. Mechanistically, KLF10 targets the VEGFR2 promoter in PACs which may underlie the observed effects. These findings may be clinically relevant because KLF10 expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-ß1 signaling and KLF10 as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states.

Noncanonical K27-linked polyubiquitination of TIEG1 regulates Foxp3 expression and tumor growth.

Earlier, we demonstrated the essential role of Kruppel-like transcription factor, TIEG1, in TGF-ß-induced regulatory T cell (Treg) development. In this article, we demonstrate that IL-6, which promotes Th17 development, abrogated TIEG1 nuclear translocation and inhibited TGF-ß-induced Treg development. Tyrosine kinase Tyk2-mediated phosphorylation of TIEG1 at Tyr179 promoted noncanonical K-27-linked polyubiquitination, which inhibited TIEG1 nuclear translocation. To test the role of TIEG1-regulated Treg/Th17 development in antitumor immunity, we analyzed TRAMP-C2 tumor growth in TIEG1(-/-) mice. The defective Treg development and elevated Th17 response resulted in enhanced immune reactivity in the tumor and inhibition of TRAMP-C2 tumor growth in TIEG1(-/-) mice. Thus, our results uncovered a novel regulatory mechanism that modulates Tregs and may regulate tumor progression.

Development, characterization, and applications of a novel estrogen receptor beta monoclonal antibody.

The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity. However, much less is known about the role of ERß and its relevance remains unclear due to the publication of conflicting reports. Here, we provide evidence that much of this controversy may be explained by variability in antibody sensitivity and specificity and describe the development, characterization, and potential applications of a novel monoclonal antibody targeting full-length human ERß and its splice variant forms. Specifically, we demonstrate that a number of commercially available ERß antibodies are insensitive for ERß and exhibit significant cross-reaction with ERα. However, our newly developed MC10 ERß antibody is shown to be highly specific and sensitive for detection of full-length ERß and its variant forms. Strong and variable staining patterns for endogenous levels of ERß protein were detected in normal human tissues and breast tumors using the MC10 antibody. Importantly, ERß was shown to be expressed in a limited cohort of both ERα positive and ERα negative breast tumors. Taken together, these data demonstrate that the use of poorly validated ERß antibodies is likely to explain much of the controversy in the field with regard to the biological relevance of ERß in breast cancer. The use of the MC10 antibody, in combination with highly specific antibodies targeting only full-length ERß, is likely to provide additional discriminatory features in breast cancers that may be useful in predicting response to therapy.

TGFß-inducible early gene-1 (TIEG1) mutations in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease. A recent study showed that male KLF10-encoded TGFß Inducible Early Gene-1 knock-out mice (TIEG-/-) develop HCM with 13-fold up-regulation of PTTG1-encoded pituitary tumor-transforming gene 1. We hypothesized TIEG1 could be a novel candidate gene in the pathogenesis of genotype negative HCM in humans, possibly through a loss of its repression on PTTG1 expression. A cohort of 923 unrelated patients from two independent HCM centers was analyzed for mutations in TIEG's four translated exons using DHPLC and direct DNA-sequencing. Site directed mutagenesis was performed to clone novel variants. The effect of TIEG1 mutations on SMAD7 and PTTG1 promoters was studied using transient transfection and luciferase-assays. Altered expression of PTTG1 in cardiac tissue was studied by immunohistochemistry (IHC) to determine levels of PTTG1 protein in hypertrophic diseases. Six novel TIEG1 missense mutations were discovered in six patients (two males/four females, mean age at diagnosis 56.2±23 years, MLVWT 20.8±4 mm). Compared to WT TIEG1, five TIEG1 mutants significantly increased PTTG1 promoter function similar to TIEG1-/--mice. By IHC, PTTG1-protein expression was significantly increased in multiple models of hypertrophic cardiac disease, including TIEG1-mutation positive HCM compared to normal hearts. This is the first article to associate mutations in TIEG1 to human disease with the discovery of six novel, HCM-associated variants. Functional assays suggest a role for PTTG1 in the pathogenesis of TIEG1-mediated HCM. Up-regulation of PTTG1 seems to be a common pathway in hypertrophic heart disease, including TIEG1-mediated HCM.

TIEG1-null tenocytes display age-dependent differences in their gene expression, adhesion, spreading and proliferation properties.

The remodeling of extracellular matrix is a crucial mechanism in tendon development and the proliferation of fibroblasts is a key factor in this process. The purpose of this study was to further elucidate the role of TIEG1 in mediating important tenocyte properties throughout the aging process. Wildtype and TIEG1 knockout tenocytes adhesion, spreading and proliferation were characterized on different substrates (fibronectin, collagen type I, gelatin and laminin) and the expression levels of various genes known to be involved with tendon development were analyzed by RT-PCR. The experiments revealed age-dependent and substrate-dependent properties for both wildtype and TIEG1 knockout tenocytes. Taken together, our results indicate an important role for TIEG1 in regulating tenocytes adhesion, spreading, and proliferation throughout the aging process. Understanding the basic mechanisms of TIEG1 in tenocytes may provide valuable information for treating multiple tendon disorders.

Estrogen receptor-beta sensitizes breast cancer cells to the anti-estrogenic actions of endoxifen.

INTRODUCTION: We have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha (ERα). However, the relevance of ERß in mediating endoxifen action has yet to be explored. Here, we characterize the molecular actions of endoxifen in breast cancer cells expressing ERß and examine its effectiveness as an anti-estrogenic agent in these cell lines. METHODS: MCF7, Hs578T and U2OS cells were stably transfected with full-length ERß. ERß protein stability, dimer formation with ERα and expression of known ER target genes were characterized following endoxifen exposure. The ability of various endoxifen concentrations to block estrogen-induced proliferation of MCF7 parental and ERß-expressing cells was determined. The global gene expression profiles of these two cell lines was monitored following estrogen and endoxifen exposure and biological pathway analysis of these data sets was conducted to identify altered cellular processes. RESULTS: Our data demonstrate that endoxifen stabilizes ERß protein, unlike its targeted degradation of ERα, and induces ERα/ERß heterodimerization in a concentration dependent manner. Endoxifen is also shown to be a more potent inhibitor of estrogen target genes when ERß is expressed. Additionally, low concentrations of endoxifen observed in tamoxifen treated patients with deficient CYP2D6 activity (20 to 40 nM) markedly inhibit estrogen-induced cell proliferation rates in the presence of ERß, whereas much higher endoxifen concentrations are needed when ERß is absent. Microarray analyses reveal substantial differences in the global gene expression profiles induced by endoxifen at low concentrations (40 nM) when comparing MCF7 cells which express ERß to those that do not. These profiles implicate pathways related to cell proliferation and apoptosis in mediating endoxifen effectiveness at these lower concentrations. CONCLUSIONS: Taken together, these data demonstrate that the presence of ERß enhances the sensitivity of breast cancer cells to the anti-estrogenic effects of endoxifen likely through the molecular actions of ERα/ß heterodimers. These findings underscore the need to further elucidate the role of ERß in the biology and treatment of breast cancer and suggest that the importance of pharmacologic variation in endoxifen concentrations may differ according to ERß expression.

Kruppel-like factor 10 protects against acute viral myocarditis by negatively regulating cardiac MCP-1 expression.

Viral myocarditis (VMC) is a cardiac disease associated with myocardial inflammation and injury induced by virus infection. Cardiomyocytes have recently been regarded as key players in eliciting and modulating inflammation within the myocardium. Kruppel-like factor 10 (KLF10) is a crucial regulator of various pathological processes and plays different roles in a variety of diseases. However, its role in VMC induced by coxsackievirus B3 (CVB3) infection remains unknown. In this study, we report that cardiac KLF10 confers enhanced protection against viral myocarditis. We found that KLF10 expression was downregulated upon CVB3 infection. KLF10 deficiency enhanced cardiac viral replication and aggravated VMC progress. Bone marrow chimera experiments indicated that KLF10 expression in nonhematopoietic cells was involved in the pathogenesis of VMC. We further identified MCP-1 as a novel target of KLF10 in cardiomyocytes, and KLF10 cooperated with histone deacetylase 1 (HDAC1) to negatively regulate MCP-1 expression by binding its promoter, leading to activation of MCP-1 transcription and recruitment of Ly6Chigh monocytes/macrophages into the myocardium. This novel mechanism of MCP-1 regulation by KLF10 might provide new insights into the pathogenesis of VMC and a potential therapeutic target for VMC.

Kruppel-like factor KLF10 targets transforming growth factor-beta1 to regulate CD4(+)CD25(-) T cells and T regulatory cells.

CD4(+)CD25(+) regulatory T cells (T regs) play a major role in the maintenance of self-tolerance and immune suppression, although the mechanisms controlling T reg development and suppressor function remain incompletely understood. Herein, we provide evidence that Kruppel-like factor 10 (KLF10/TIEG1) constitutes an important regulator of T regulatory cell suppressor function and CD4(+)CD25(-) T cell activation through distinct mechanisms involving transforming growth factor (TGF)-beta1 and Foxp3. KLF10 overexpressing CD4(+)CD25(-) T cells induced both TGF-beta1 and Foxp3 expression, an effect associated with reduced T-Bet (Th1 marker) and Gata3 (Th2 marker) mRNA expression. Consistently, KLF10(-/-) CD4(+)CD25(-) T cells have enhanced differentiation along both Th1 and Th2 pathways and elaborate higher levels of Th1 and Th2 cytokines. Furthermore, KLF10(-/-) CD4(+)CD25(-) T cell effectors cannot be appropriately suppressed by wild-type T regs. Surprisingly, KLF10(-/-) T reg cells have reduced suppressor function, independent of Foxp3 expression, with decreased expression and elaboration of TGF-beta1, an effect completely rescued by exogenous treatment with TGF-beta1. Mechanistic studies demonstrate that in response to TGF-beta1, KLF10 can transactivate both TGF-beta1 and Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-intrinsic control of T cell activation. Finally, KLF10(-/-) CD4(+)CD25(-) T cells promoted atherosclerosis by approximately 2-fold in ApoE(-/-)/scid/scid mice with increased leukocyte accumulation and peripheral pro-inflammatory cytokines. Thus, KLF10 is a critical regulator in the transcriptional network controlling TGF-beta1 in both CD4(+)CD25(-) T cells and T regs and plays an important role in regulating atherosclerotic lesion formation in mice.

2-methoxyestradiol-mediated anti-tumor effect increases osteoprotegerin expression in osteosarcoma cells.

Osteosarcoma is a bone tumor that frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, induces cell cycle arrest and cell death in human osteosarcoma cells. To investigate whether the osteoprotegrin (OPG) protein plays a role in 2-ME actions, we studied the effect of 2-ME treatment on OPG gene expression in human osteosarcoma cells. 2-ME treatment induced OPG gene promoter activity and mRNA levels. Also, Western blot analysis showed that 2-ME treatment increased OPG protein levels in MG63, KHOS, 143B and LM7 osteosarcoma cells by 3-, 1.9-, 2.8-, and 2.5-fold, respectively, but did not affect OPG expression in normal bone cells. In addition, increases in OPG protein levels were observed in osteosarcoma cell culture media after 3 days of 2-ME treatment. The effect of 2-ME on osteosarcoma cells was ligand-specific as parent estrogen, 17beta-estradiol and a tumorigenic estrogen metabolite, 16alpha-hydroxyestradiol, which do not affect osteosarcoma cell cycle and cell death, had no effect on OPG protein expression. Furthermore, co-treating osteosarcoma cells with OPG protein did not further enhance 2-ME-mediated anti-tumor effects. OPG-released in 2-ME-treated cultures led to an increase in osteoblastic activity and a decrease in osteoclast number, respectively. These findings suggest that OPG is not directly involved in 2-ME-mediated anti-proliferative effects in osteosarcoma cells, but rather participates in anti-resorptive functions of 2-ME in bone tumor environment.

Histone demethylase JARID1B/KDM5B is a corepressor of TIEG1/KLF10.

JARID1B/KDM5B (jumonji AT-rich interactive domain 1B/lysine-specific demethylase 5B) is an enzyme that efficiently removes methyl residues from trimethylated lysine 4 on histone H3, a pivotal mark for active chromatin. TIEG1/KLF10 (transforming growth factor-ß inducible early gene-1/Krüppel-like transcription factor 10) is a zinc-finger transcription factor that is involved in bone metabolism and exerts antiproliferative activity. Here, we found that TIEG1 interacts with JARID1B. In particular, the repression domains of TIEG1 bind to the C-terminus of JARID1B. Moreover, overexpression of JARID1B augments TIEG1 to repress transcription of Smad7, an inhibitor of the TGF-ß (transforming growth factor-ß) signaling pathway. Conversely, JARID1B knock-down leads to increased Smad7 mRNA levels. Thus, TIEG1 and JARID1B may cooperate to suppress tumorigenesis by enhancing TGF-ß signaling. Notably, both TIEG1 and JARID1B are downregulated in melanomas, suggesting that they indeed cooperate physiologically. In conclusion, JARID1B is the first TIEG1 corepressor identified, explaining how TIEG1 represses transcription through inducing histone H3 lysine 4 demethylation, which may be important for TIEG1 function in both normal and cancer cells.

Retinoblastoma binding protein-1 (RBP1) is a Runx2 coactivator and promotes osteoblastic differentiation.

BACKGROUND: Numerous transcription factors are involved in the establishment and maintenance of the osteoblastic phenotype, such as Runx2, osterix and Dlx5. The transcription factor retinoblastoma binding protein-1 (RBP1) was recently identified as an estrogen regulated gene in an osteosarcoma cell model. Since the function of RBP1 in osteoblastic differentiation and mineralization is unknown, we investigated the role of RBP1 in these processes. METHODS: To create a cell model with suppressed RBP1 expression, primary calvarial osteoblasts were infected with a shRNA lentiviral vector specific for mouse RBP1 (CalOB-DeltaRBP1) or a scrambled control shRNA lentivirus (CalOB-Control). Stable cell lines were generated and their mineralization potential was determined using osteoblastic differentiation medium, Alizarin Red staining, and quantitative PCR (QPCR) analyses. Runx2 coactivation by RBP1 was determined through the use of transient transfection assays. RESULTS: Stable expression of the RBP1 shRNA lentivirus in CalOB-DeltaRBP1 cells resulted in a 65-70% suppression of RBP1 expression. Osteoblastic mineralization assays demonstrated that suppression of RBP1 results in a potent delay in osteoblastic nodule formation in the CalOB-DeltaRBP1 cells with a concomitant decrease in the expression of the osteogenic transcription factors Runx2 and osterix, along with decreases in BMP2, alkaline phosphatase, osteocalcin and bone sialoprotein. Regulation of Runx2 expression by RBP1 was shown to be mediated through the proximal P2 Runx2 promoter. Furthermore, RBP1 was demonstrated to be a potent coactivator of Runx2 transcriptional activity on two known Runx2 reporter constructs. These data suggest that the expression and activity of Runx2 is critically dependent on the presence of RBP1. CONCLUSIONS: This study is the first to demonstrate that RBP1 is an important mediator of the osteoblastic phenotype and clearly defines RBP1 as a novel transcription factor involved in the well known Runx2-osterix transcriptional cascade. As such, the effects of RBP1 on these processes are mediated through both regulation of Runx2 expression and transcriptional activity.

Estrogen receptor beta isoform-specific induction of transforming growth factor beta-inducible early gene-1 in human osteoblast cells: an essential role for the activation function 1 domain.

The estrogen receptors (ER) alpha and beta are important ligand-mediated transcription factors known to play significant biological roles in numerous tissues including bone. Despite the high homology shared by these receptors, recent studies have suggested that their function is largely unique. Although these receptors have been studied in detail for more than a decade, little data exist concerning the mechanisms by which these two proteins regulate distinct sets of genes. Using the TGFbeta-inducible early gene-1 (TIEG) as a model, we demonstrate that TIEG is rapidly induced in response to estrogen in osteoblasts by ERbeta, but not ERalpha. We have identified the regulatory elements utilized by ERbeta and have demonstrated that ERbeta recruits steroid receptor coactivator (SRC)1 and SRC2 to this regulatory region. Additionally, deletion of the ERbeta-activation function 1 (AF1) domain drastically decreases the estrogen induction of TIEG. Through the use of chimeric receptors, we have demonstrated that the AF1 domain of ERbeta is responsible for recruiting SRC1 and SRC2 and inducing the expression of TIEG in osteoblasts. Finally, SRC1, but not SRC2, is essential for TIEG induction by ERbeta. Overall, these data demonstrate that the estrogen induction of TIEG is ERbeta specific and that the AF1 domain of ERbeta confers this specificity. Finally, a novel and important role for ERbeta's AF1 is implicated in the recruitment of specific coactivators, suggesting that the AF1 may play a significant role in conferring the differences in regulation of gene expression by these two receptors.

TIEG1-NULL OSTEOCYTES DISPLAY DEFECTS IN THEIR MORPHOLOGY, DENSITY AND SURROUNDING BONE MATRIX.

Through the development of TGFß-inducible early gene-1 (TIEG1) knockout (KO) mice, we have demonstrated that TIEG1 plays an important role in osteoblast-mediated bone mineralization, and in bone resistance to mechanical strain. To further investigate the influence of TIEG1 in skeletal maintenance, osteocytes were analyzed by transmission electron microscopy using TIEG1 KO and wild-type mouse femurs at one, three and eight months of age. The results revealed an age-dependent change in osteocyte surface and density, suggesting a role for TIEG1 in osteocyte development. Moreover, there was a decrease in the amount of hypomineralized bone matrix surrounding the osteocytes in TIEG1 KO mice relative to wild-type controls. While little is known about the function or importance of this hypomineralized bone matrix immediately adjacent to osteocytes, this study reveals significant differences in this bone microenvironment and suggests that osteocyte function may be compromised in the absence of TIEG1 expression.

Bone growth and turnover in progesterone receptor knockout mice.

The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and microcomputed tomography analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 wk of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain, and tibia longitudinal bone growth were normal in PRKO mice. In contrast, total, cancellous, and cortical bone mass were increased in the humerus of 12-wk-old PRKO mice, whereas cortical and cancellous bone mass in the tibia was normal. At 26 wk of age, cancellous bone area in the proximal tibia metaphysis of PRKO mice was 153% greater than age matched wild-type mice. The improved cancellous bone balance in 6-month-old PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice is not essential for bone growth and turnover. However, at some skeletal sites, PR signaling attenuates the accumulation of cortical and cancellous bone mass during adolescence.

Rcl is a novel ETV1/ER81 target gene upregulated in breast tumors.

ETV1 (ER81) is a transcription factor that can be activated by HER2/Neu, a proto-oncoprotein often overexpressed in metastatic breast tumors. Here, we demonstrate that ETV1 downregulation suppresses proliferation of HER2/Neu-positive MDA-MB-231 breast cancer cells in vitro and tumor formation in vivo, proving for the first time the existence of a critical role of ETV1 in breast cancer cell physiology. A screen for novel ETV1 target genes hinted at Rcl, an enzyme involved in nucleotide metabolism. To characterize the human Rcl gene, we cloned its promoter and found that ETV1 and HER2/Neu cooperated in activating the Rcl promoter, whereas a dominant-negative ETV1 molecule suppressed the Rcl promoter. Moreover, ETV1 and HER2/Neu synergized to upregulate the endogenous Rcl gene. ETV1 also bound to the Rcl promoter in vivo, indicating that Rcl is a bona fide target gene of ETV1. Hybridization of Rcl cDNA to a breast cancer array revealed that Rcl is overexpressed in approximately 40% of all breast tumors. Importantly, its expression significantly escalates with increasing tumor grade, strongly implicating that Rcl contributes to breast tumorigenesis. Since joint overexpression of Rcl with vascular endothelial growth factor, another target gene of ETV1, has been shown to induce tumor formation, Rcl may be one crucial effector of ETV1 and HER2/Neu in breast tumors. Furthermore, given its expression pattern and enzymatic function in nucleotide metabolism, Rcl presents itself as a novel target in breast cancer therapy via modulation of its activity by small molecule drugs.

Loss of ERE binding activity by estrogen receptor-alpha alters basal and estrogen-stimulated bone-related gene expression by osteoblastic cells.

Estrogen receptor (ER)-alpha can signal either via estrogen response element (ERE)-mediated pathways or via alternate pathways involving protein-protein or membrane signaling. We previously demonstrated that, as compared to wild type (WT) controls, mice expressing a mutant ER-alpha lacking the ability to bind EREs (non-classical estrogen receptor knock-in (NERKI)) display significant impairments in the skeletal response to estrogen. To elucidate the mechanism(s) underlying these in vivo deficits, we generated U2OS cells stably expressing either WT ER-alpha or the NERKI receptor. Compared to cells transfected with the control vector, stable expression of ER-alpha, even in the absence of E2, resulted in an increase in mRNA levels for alkaline phosphatase (AP, by 400%, P < 0.01) and a decrease in mRNA levels for insulin growth factor-I (IGF-I) (by 65%, P < 0.001), with no effects on collagen I (col I) or osteocalcin (OCN) mRNA levels. By contrast, stable expression of the NERKI receptor resulted in the suppression of mRNA levels for AP, col I, OCN, and IGF-I (by 62, 89, 60, and 70%, P < 0.001). While E2 increased mRNA levels of AP, OCN, col I, and IGF-I in ER-alpha cells, E2 effects in the NERKI cells on AP and OCN mRNA levels were attenuated, with a trend for E2 to inhibit col I mRNA levels. In addition, E2 had no effects on IGF-I mRNA levels in NERKI cells. Collectively, these findings indicate that ERE signaling plays a significant role in mediating effects of estrogen on osteoblastic differentiation markers and on IGF-I mRNA levels.

TIEG1 null mouse-derived osteoblasts are defective in mineralization and in support of osteoclast differentiation in vitro.

Transforming growth factor beta-inducible early gene 1 (TIEG1) is a member of the Kruppel-like transcription factor family. To understand the physiological role of TIEG1, we generated TIEG(-/-) (null) mice and found that the TIEG(-/-) mice had increased osteoblast numbers with no increased bone formation parameters. However, when calvarial osteoblasts (OBs) were isolated from neonatal TIEG(-/-) and TIEG(+/+) mice and cultured in vitro, the TIEG(-/-) cells displayed reduced expression of important OB differentiation markers. When the OBs were differentiated in vitro by treatment with bone morphogenic protein 2, the OBs from TIEG(+/+) calvaria displayed several mineralized nodules in culture, whereas those from TIEG(-/-) mice showed no nodules. To characterize the OBs' ability to support osteoclast differentiation, the OBs from TIEG(+/+) and TIEG(-/-) mice were cultured with marrow and spleen cells from TIEG(+/+) mice. Significantly fewer osteoclasts developed when TIEG(-/-) OBs were used to support osteoclast differentiation than when TIEG(+/+) OBs were used. Examination of gene expression in the TIEG(-/-) OBs revealed decreased RANKL and increased OPG expression compared to TIEG(+/+) OBs. The addition of RANKL to these cocultures only partially restored the ability of TIEG(-/-) OBs to support osteoclast differentiation, whereas M-CSF alone or combined with RANKL had no additional effect on osteoclast differentiation. We conclude from these data that TIEG1 expression in OBs is critical for both osteoblast-mediated mineralization and osteoblast support of osteoclast differentiation.

WOUND-HEALING PROPERTIES OF TRANSFORMING GROWTH FACTOR ß (TGF-ß) INDUCIBLE EARLY GENE 1 (TIEG1) KNOCKOUT MICE.

Transforming growth factor beta (TGF-ß) has a broad effect on wound healing, but many questions remain about the regulation of TGF-ß during the healing process. TGF-ß inducible early gene 1 (TIEG1) is a primary response gene for TGF-ß that controls the activities of the TGF-ß/Smad pathway, the primary TGF-ß signaling pathway. The purpose of this study was to investigate the role of TIEG1 in cutaneous wound healing using TIEG1 knockout mice. The wound healing in TIEG1 knockout mice and wild-type controls was evaluated by wound breaking strength, Western blot, and histology at postoperative days 3, 7, and 14. Although re-epithelialization of both groups was similarly complete at day 7, the TIEG1 knockout mice had a significantly lower wound breaking strength than the controls at postoperative day 14. These results suggest that TIEG1 expression may be an important factor involved in the initiation and support of normal cutaneous wound healing.