Growth Differentiation Factor 7 promotes multiple-lineage differentiation in tenogenic cultures of mesenchymal stem cells.

The repair of the tendon-bone interface, which is composed of tendon, fibrocartilage, and bony attachment, remains a clinical challenge. The application of mesenchymal stem cells (MSCs), collagen-rich extracellular matrix (ECMs), as well as growth factors, has the potential to regenerate this special multiple-tissue structure through the so-called biological augmentation. We present here an in vitro tendon regeneration model with C3H10T1/2 cells cultured on Collagen I matrix and evaluated the lineage determination effects of Growth Differentiation Factor 7 (GDF-7). We found that besides tenogenic effect, GDF-7 also stimulates the expression of osteoblastic as well as adipocytic genes. Our results indicate that GDF-7 might be a promising growth factor for regeneration of the tendon-bone interface due to its multiple-lineage stimulating effects. However, the side effect on adipogenic differentiation should be of concern, as it is a known risk factor for repair failures.

ERα and ERß Homodimers in the Same Cellular Context Regulate Distinct Transcriptomes and Functions.

The two estrogen receptors ERα and ERß are nuclear receptors that bind estrogen (E2) and function as ligand-inducible transcription factors. They are homologues and can form dimers with each other and bind to the same estrogen-response element motifs in the DNA. ERα drives breast cancer growth whereas ERß has been reported to be anti-proliferative. However, they are rarely expressed in the same cells, and it is not fully investigated to which extent their functions are different because of inherent differences or because of different cellular context. To dissect their similarities and differences, we here generated a novel estrogen-dependent cell model where ERα homodimers can be directly compared to ERß homodimers within the identical cellular context. By using CRISPR-cas9 to delete ERα in breast cancer MCF7 cells with Tet-Off-inducible ERß expression, we generated MCF7 cells that express ERß but not ERα. MCF7 (ERß only) cells exhibited regulation of estrogen-responsive targets in a ligand-dependent manner. We demonstrated that either ER was required for MCF7 proliferation, but while E2 increased proliferation via ERα, it reduced proliferation through a G2/M arrest via ERß. The two ERs also impacted migration differently. In absence of ligand, ERß increased migration, but upon E2 treatment, ERß reduced migration. E2 via ERα, on the other hand, had no significant impact on migration. RNA sequencing revealed that E2 regulated a transcriptome of around 800 genes via each receptor, but over half were specific for either ERα or ERß (417 and 503 genes, respectively). Functional gene ontology enrichment analysis reinforced that E2 regulated cell proliferation in opposite directions depending on the ER, and that ERß specifically impacted extracellular matrix organization. We corroborated that ERß bound to cis-regulatory chromatin of its unique proposed migration-related direct targets ANXA9 and TFAP2C. In conclusion, we demonstrate that within the same cellular context, the two ERs regulate cell proliferation in the opposite manner, impact migration differently, and each receptor also regulates a distinct set of target genes in response to E2. The developed cell model provides a novel and valuable resource to further complement the mechanistic understanding of the two different ER isoforms.

Blocking Fra-1 sensitizes triple-negative breast cancer to PARP inhibitor.

The AP-1 member Fra-1 is overexpressed in TNBC and plays crucial roles in tumor progression and treatment resistance. In a previous large-scale screen, we identified PARP1 to be among 118 proteins that interact with endogenous chromatin-bound Fra-1 in TNBC cells. PARP1 inhibitor (olaparib) is currently in clinical use for treatment of BRCA-mutated TNBC breast cancer. Here, we demonstrate that the Fra-1-PARP1 interaction impacts the efficacy of olaparib treatment. We show that PARP1 interacts with and downregulates Fra-1, thereby reducing AP-1 transcriptional activity. Olaparib treatment, or silencing of PARP1, consequently, increases Fra-1 levels and enhances its transcriptional activity. Increased Fra-1 can have adverse effect, including treatment resistance. We also found that a large fraction of PARP1-regulated genes was dependent on Fra-1. We show that by inhibiting Fra-1/AP-1, non-BRCA-mutated TNBC cells can become sensitized to olaparib treatment. We identify that high PARP1 expression is indicative of a poor clinical outcome in breast cancer patients overall (P = 0.01), but not for HER-2 positive patients. In conclusion, by exploring the functionality of the Fra-1 and PARP1 interaction, we propose that targeting Fra-1 could serve as a combinatory therapeutic approach to improve olaparib treatment outcome for TNBC patients.

Endogenous interaction profiling identifies DDX5 as an oncogenic coactivator of transcription factor Fra-1.

Fra-1, a member of the activator protein 1 (AP-1) family, is overexpressed in triple-negative breast cancer (TNBC) and plays crucial roles in tumor growth. Here we report the identification of 118 proteins interacting with endogenous chromatin-bound Fra-1 in TNBC cells, highlighting DDX5 as the most enriched Fra-1-interacting protein. DDX5, a previously unrecognized protein in the Fra-1 transcriptional network, shows extensive overlap with Fra-1 cistrome and transcriptome that are highly associated with the TNBC cell growth. We provide evidence that DDX5 expression enhances Fra-1 transcriptional activity and potentiates Fra-1-driven cell proliferation. Furthermore, we show that the DDX5 target gene signature predicts poor clinical outcome in breast cancer patients. DDX5 protein level was higher in triple-negative basal-like tumors than in non-basal-like tumors, including luminal A, luminal B, and HER2-enriched subtypes. Collectively, by combining proteomic and genomic approaches we reveal a role for DDX5 as a regulatory protein of Fra-1 signaling and suggest DDX5 as a potential therapeutic target for TNBC.

Expression of the three components of linear ubiquitin assembly complex in breast cancer.

Proteins belonging to the linear ubiquitin assembly complex (LUBAC) are believed to be important in tumorigenesis. LUBAC has been demonstrated to be composed of RBCK1, RNF31 and SHARPIN. The aim of this study was to explore all members of the LUBAC complex as novel biomarkers in breast cancer. We have already reported that RNF31 mRNA levels are higher in breast cancer samples compared to adjacent non-tumor tissue. In this study we extend these findings by demonstrating that the mRNA levels of RBCK1 and SHARPIN are also higher in tumors compared to adjacent non-tumor tissue in the same cross sectional study of samples (p < 0.001). In addition, up-regulated mRNA expression of all three members of the LUBAC complex displayed high predictive value in distinguishing tumor tissues from adjacent non-tumor tissue as determined by ROC curve analysis. Furthermore, we investigated whether there is an association between the mRNA and protein expression levels of RBCK1, RNF31 and SHARPIN and clinicopathological parameters including estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2) status and found that RNF31 protein is significantly higher in ERalpha-negative tumors than ERalpha-positive tumors (p = 0.034). Collectively, our findings indicate that up-regulated mRNA expression of RNF31, RBCK1 and SHARPIN could potentially be diagnostic biomarkers of breast cancer and RNF31 might be a drug target for ERalpha-negative breast cancers.

c-Jun/AP-1 overexpression reprograms ERα signaling related to tamoxifen response in ERα-positive breast cancer.

A critical mechanism that has been proposed for transcription regulation by estrogen receptor α (ER) is the tethering of ER to DNA via other transcription factors, such as AP-1. However, genome-wide assessment of the overlap in chromatin binding repertoires of these two transcription factors has not been reported. Here, we show that the AP-1 transcription factor c-Jun interacts with ER and that c-Jun chromatin binding shows extensive overlap with ER binding at the global level. Further, we show that c-Jun overexpression reprograms ER chromatin binding and modulates ER-mediated gene regulation. Our data are consistent with a mechanism where estrogen/ER-dependent crosstalk with AP-1 at the transcriptional level is mediated through the tethering of ER to DNA bound AP-1. Additionally, in our system c-Jun overexpression causes reduced sensitivity to tamoxifen in ER+ breast cancer cells. Integrated cistrome, transcriptome, and clinical data reveal TGFBI as a candidate gene which may confer tamoxifen resistance by ER and AP-1 crosstalk. Further, we show that TGFBI expression is elevated in breast cancer compared to normal breast. Together, our data provide a novel genome-wide footprint of ER and AP-1 crosstalk and suggest AP-1 and TGFBI signaling as potential therapeutic targets in AP-1-overexpressing ER-positive breast tumors.

Estrogen receptor ß2 induces proliferation and invasiveness of triple negative breast cancer cells: association with regulation of PHD3 and HIF-1α.

The two estrogen receptor (ER) subtypes, ERα and ERß, belong to the nuclear receptor superfamily. The human ERß variant ERß2 is proposed to be expressed at higher levels than ERß1 in many breast tumors and it has been suggested that ERß2, in contrast to ERß1, is associated with aggressive phenotypes of various cancers. However, the role of endogenous ERß2 in breast cancer cells remains elusive. In this study, we identified that triple negative breast cancer (TNBC) cell lines express endogenous ERß2, but not ERα or ERß1. This allows novel studies of endogenous ERß2 functions independent of ERα and ERß1. We show that overexpression of ERß2 in TNBC cells increased whereas knockdown of endogenous ERß2 decreased cell proliferation and cell invasion. To elucidate the molecular mechanism responsible for these cellular phenotypes, we assayed ERß2 dependent global gene expression profiles. We show that ERß2 decreases prolyl hydroxylase 3 (PHD3) gene expression and further show that this is associated with increased hypoxia inducible factor 1α (HIF-1α) protein levels, thus providing a possible mechanism for the invasive phenotype. These results are further supported by analysing the expression of ERß2 and PHD3 in breast tumor samples where a negative correlation between ERß2 and PHD3 expression was observed. Together, we demonstrate that ERß2 has an important role in enhancing cell proliferation and invasion, beyond modulation of ERß and ERß1 signalling which might contribute to the invasive characteristics of TNBC. The invasive phenotype could potentially be mediated through transcriptional repression of PHD3 and increased HIF-1α protein levels.

Estrogen Receptor α Promotes Breast Cancer by Reprogramming Choline Metabolism.

Estrogen receptor α (ERα) is a key regulator of breast growth and breast cancer development. Here, we report how ERα impacts these processes by reprogramming metabolism in malignant breast cells. We employed an integrated approach, combining genome-wide mapping of chromatin-bound ERα with estrogen-induced transcript and metabolic profiling, to demonstrate that ERα reprograms metabolism upon estrogen stimulation, including changes in aerobic glycolysis, nucleotide and amino acid synthesis, and choline (Cho) metabolism. Cho phosphotransferase CHPT1, identified as a direct ERα-regulated gene, was required for estrogen-induced effects on Cho metabolism, including increased phosphatidylcholine synthesis. CHPT1 silencing inhibited anchorage-independent growth and cell proliferation, also suppressing early-stage metastasis of tamoxifen-resistant breast cancer cells in a zebrafish xenograft model. Our results showed that ERα promotes metabolic alterations in breast cancer cells mediated by its target CHPT1, which this study implicates as a candidate therapeutic target. Cancer Res; 76(19); 5634-46. ©2016 AACR.

Induction of USP17 by combining BET and HDAC inhibitors in breast cancer cells.

Members of the bromodomain and extra-C terminal (BET) domain protein family and the histone deacetylase (HDAC) enzyme family regulate the expression of important oncogenes and tumor suppressor genes. Here we show that the BET inhibitor JQ1 inhibits proliferation and induces apoptosis of both triple negative and estrogen receptor positive breast cancer cells. Consistent with the critical role of histone acetylation in the regulation of gene expression, treatment with JQ1 or the HDAC inhibitor mocetinostat was associated with global changes in gene expression resulting in suppression of genes involved in cell-cycle regulation. Combining JQ1 with mocetinostat, further decreased cell viability. This synergistic effect was associated with increased suppression of genes essential for cell-cycle progression. Furthermore, we detected dramatic increase in the expression of several members of the ubiquitin-specific protease 17 (USP17) family of deubiquitinating enzymes in response to the combination treatment. Increased expression of USP17 enzymes were able to attenuate the Ras/MAPK pathway causing decrease in cell viability, while, siRNA mediated depletion of USP17 significantly decreased cytotoxicity after the combination treatment. In conclusion, our study demonstrates that co-treatment with BET inhibitors and HDAC inhibitors reduces breast cancer cell viability through induction of USP17.

Estrogen receptor beta in breast cancer.

Estrogen is essential for growth and development of the mammary glands and has been associated with the promotion and growth of breast cancer and in line with this, most human breast cancers are initially estrogen-dependent and undergo regression when deprived of their supporting hormone. Estrogen exerts many of its effects via two nuclear estrogen receptors (ERs), ERα and ERß. The discovery of a second ER, ERß, demanded a full re-evaluation of estrogen action in all target tissues and different estrogen associated diseases, including human breast cancer. However, despite over 15 years of research, the exact role, if any, of ERß in human breast cancer remains elusive. The main challenges now are to develop highly selective anti-ERß antibodies that are applied to large well characterized human breast cancer samples to validate their diagnostic potential and to explore ERß-selective agonists in animal models of breast cancer to validate their therapeutic potential.

Diclofenac and triamcinolone acetonide impair tenocytic differentiation and promote adipocytic differentiation of mesenchymal stem cells.

BACKGROUND: Tendinopathies are often empirically treated with oral/topical nonsteroidal anti-inflammatory medications and corticosteroid injections despite their unclear effects on tendon regeneration. Recent studies indicate that tendon progenitors exhibit stem cell-like properties, i.e., differentiation to osteoblasts, adipocytes, and chondrocytes, in addition to tenocytes. Our present study aims at understanding the effects of triamcinolone acetonide and diclofenac on tenocytic differentiation of mesenchymal stem cells. METHODS: The murine fibroblast C3H10T1/2 cell line was induced to tenocytic differentiation by growth differentiation factor-7. Cell proliferation and differentiation with the exposure of different concentrations of triamcinolone acetonide and diclofenac were measured by WST-1 assay and real-time polymerase chain reaction analysis, respectively. RESULTS: Cell proliferation was decreased in a concentration-dependent manner when exposed to triamcinolone acetonide and diclofenac. In addition to tenocytic differentiation, adipocyte formation was observed, both at gene expression and microscopic level, when the cells were exposed to triamcinolone acetonide or high concentrations of diclofenac. CONCLUSIONS: Our results indicate that triamcinolone acetonide and diclofenac might alter mesenchymal stem cell differentiation in a nonfavorable way regarding tendon regeneration; therefore, these medications should be used with more caution clinically.

MicroRNA-regulated gene networks during mammary cell differentiation are associated with breast cancer.

MicroRNAs (miRNAs) play pivotal roles in stem cell biology, differentiation and oncogenesis and are of high interest as potential breast cancer therapeutics. However, their expression and function during normal mammary differentiation and in breast cancer remain to be elucidated. In order to identify which miRNAs are involved in mammary differentiation, we thoroughly investigated miRNA expression during functional differentiation of undifferentiated, stem cell-like, murine mammary cells using two different large-scale approaches followed by qPCR. Significant changes in expression of 21 miRNAs were observed in repeated rounds of mammary cell differentiation. The majority, including the miR-200 family and known tumor suppressor miRNAs, was upregulated during differentiation. Only four miRNAs, including oncomiR miR-17, were downregulated. Pathway analysis indicated complex interactions between regulated miRNA clusters and major pathways involved in differentiation, proliferation and stem cell maintenance. Comparisons with human breast cancer tumors showed the gene profile from the undifferentiated, stem-like stage clustered with that of poor-prognosis breast cancer. A common nominator in these groups was the E2F pathway, which was overrepresented among genes targeted by the differentiation-induced miRNAs. A subset of miRNAs could further discriminate between human non-cancer and breast cancer cell lines, and miR-200a/miR-200b, miR-146b and miR-148a were specifically downregulated in triple-negative breast cancer cells. We show that miR-200a/miR-200b can inhibit epithelial-mesenchymal transition (EMT)-characteristic morphological changes in undifferentiated, non-tumorigenic mammary cells. Our studies propose EphA2 as a novel and important target gene for miR-200a. In conclusion, we present evidentiary data on how miRNAs are involved in mammary cell differentiation and indicate their related roles in breast cancer.

Estrogen receptor ß represses Akt signaling in breast cancer cells via downregulation of HER2/HER3 and upregulation of PTEN: implications for tamoxifen sensitivity.

INTRODUCTION: The inhibition of estrogen receptor (ER) α action with the ER antagonist tamoxifen is an established treatment in the majority of breast cancers. De novo or acquired resistance to this therapy is common. Expression of ERß in breast tumors has been implicated as an indicator of tamoxifen sensitivity. The mechanisms behind this observation remain largely uncharacterized. In the present study, we investigated whether ERß can modulate pathways implicated in endocrine resistance development. METHODS: T47-D and MCF-7 ERα-expressing breast cancer cells with tetracycline-regulated expression of ERß were used as a model system. Expression levels and activity of known regulators of endocrine resistance were analyzed by performing quantitative polymerase chain reaction assays, Western blot analysis and immunostaining, and sensitivity to tamoxifen was investigated by using a cell proliferation kit. RESULTS: Expression of ERß in ERα-positive T47-D and MCF-7 human breast cancer cells resulted in a decrease in Akt signaling. The active form of an upstream regulator of Akt, proto-oncogene c-ErbB-2/receptor tyrosine kinase erbB-3 (HER2/HER3) receptor dimer, was also downregulated by ERß. Furthermore, ERß increased expression of the important inhibitor of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Importantly, ERß expression increased the sensitivity of these breast cancer cells to tamoxifen. CONCLUSIONS: Our results suggest a link between expression of ERß and endocrine sensitivity by increasing PTEN levels and decreasing HER2/HER3 signaling, thereby reducing Akt signaling with subsequent effects on proliferation, survival and tamoxifen sensitivity of breast cancer cells. This study supports initiatives to further investigate whether ERß presence in breast cancer samples is an indicator for endocrine response. Current therapies in ERα-positive breast cancers aim to impair ERα activity with antagonists or by removal of endogenous estrogens with aromatase inhibitors. Data from this study could be taken as indicative for also using ERß as a target in selected groups of breast cancer.

Expression of estrogen receptor beta increases integrin alpha1 and integrin beta1 levels and enhances adhesion of breast cancer cells.

Estrogen effects on mammary gland development and differentiation are mediated by two receptors (ERalpha and ERbeta). Estrogen-bound ERalpha induces proliferation of mammary epithelial and cancer cells, while ERbeta is important for maintenance of the differentiated epithelium and inhibits proliferation in different cell systems. In addition, the normal breast contains higher ERbeta levels compared to the early stage breast cancers, suggesting that loss of ERbeta could be important in cancer development. Analysis of ERbeta-/- mice has consistently revealed reduced expression of cell adhesion proteins. As such, ERbeta is a candidate modulator of epithelial homeostasis and metastasis. Consequently, the aim of this study was to analyze estrogenic effects on adhesion of breast cancer cells expressing ERalpha and ERbeta. As ERbeta is widely found in breast cancer but not in cell lines, we used ERalpha positive T47-D and MCF-7 human breast cancer cells to generate cells with inducible ERbeta expression. Furthermore, the colon cancer cell lines SW480 and HT-29 were also used. Integrin alpha1 mRNA and protein levels increased following ERbeta expression. Integrin beta1-the unique partner for integrin alpha1-increased only at the protein level. ERbeta expression enhanced the formation of vinculin containing focal complexes and actin filaments, indicating a more adhesive potential. This was confirmed by adhesion assays where ERbeta increased adhesion to different extracellular matrix proteins, mostly laminin. In addition, ERbeta expression was associated to less cell migration. These results indicate that ERbeta affects integrin expression and clustering and consequently modulates adhesion and migration of breast cancer cells.

Gene expression in murine mammary epithelial stem cell-like cells shows similarities to human breast cancer gene expression.

INTRODUCTION: Mammary stem cells are bipotential and suggested to be the origin of breast cancer development, but are elusive and vaguely characterized. Breast tumors can be divided into subgroups, each one requiring specific treatment. To determine a possible association between mammary stem cells and breast cancer, a detailed characterization of the transcriptome in mammary stem cells is essential. METHODS: We have used a murine mammary epithelial stem-like cell line (HC11) and made a thorough investigation of global gene-expression changes during stepwise differentiation using dual-color comparative microarray technique. Subsequently, we have performed a cross-species comparison to reveal conserved gene expression between stem cells and subtype-specific and prognosis gene signatures, and correlated gene expression to in vivo mammary gland development. RESULTS: Our analysis of mammary stem-like and stepwise cell differentiation, and an in-depth description of our findings in a breast cancer perspective provide a unique map of the transcriptomic changes and a number of novel mammary stem cell markers. We correlate the alterations to in vivo mammary gland differentiation, and describe novel changes in nuclear receptor gene expression. Interestingly, our comparisons show that specific subtypes of breast cancers with poor prognosis and metastasizing capabilities show resemblance to stem-like gene expression. CONCLUSIONS: The transcriptional characterization of these mammary stem-like cells and their differentiation-induced gene expression patterns is here made widely accessible and provides a basis for research on mammary stem-like cells. Our comparisons suggest that some tumors are more stem-like than others, with a corresponding worse prognosis. This information would, if established, be important for treatment decisions. We also suggest several marker candidates valuable to investigate further.

Resveratrol inhibits proliferation and promotes apoptosis of osteosarcoma cells.

The phytoalexin resveratrol has been described to have chemopreventive and chemotherapeutic effects in several tumor models while its effects on osteosarcoma have not been extensively studied. Additionally, resveratrol is a potent activator of the Sirt1/Sir2 (silent information regulator 2) family of NAD-dependent deacetylases which plays a role in calorie restriction-mediated tumor suppression. In the present study, we evaluated the effect of resveratrol on growth and apoptosis in four osteosarcoma cell lines (HOS, Saos-2, U-2 OS and MG-63) and a normal human osteoblast cell line (NHOst). We found that Sirt1 protein was relatively higher expressed in the tumor cells than normal osteoblasts. Consistently, resveratrol induced apoptosis in a dose-dependent fashion in the osteosarcoma cells but had minor effect on normal osteoblasts. Also, a similar effect could be elicited by another Sirt1 activator, isonicotinamide. In addition, the pro-apoptotic effect of resveratrol could be enhanced by nutrition restriction elicited by l-asparaginase. We postulate that these effects by resveratrol are mediated via Sirt1 but further studies are needed to confirm or refute this theory.

Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSC) can differentiate into osteoblasts, adipocytes, chondrocytes and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSC into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of the nuclear protein deacetylase Sirt1 has recently been shown to decrease adipocyte development from preadipocytes via inhibition of PPARgamma2. In vitro, MSC differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also developed. In the present study we have targeted Sirt1 to control adipocyte development during differentiation of MSC into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation demonstrates an interesting alternative to PPARgamma antagonists. These results are important for the evolving field of cell-based tissue engineering, but may also be relevant in the search for new treatments of osteoporosis.

Different roles of estrogen receptors alpha and beta in the regulation of E-cadherin protein levels in a mouse mammary epithelial cell line.

Two estrogen receptors (ERalpha and ERbeta) are found throughout the mammary gland. Evidence indicates that, while ERalpha transduces proliferation signals, ERbeta opposes this effect and is necessary for epithelial differentiation. Using mouse mammary epithelial cells, we have previously shown that activation of ERbeta opposes ERalpha-induced proliferation and increases apoptosis. Furthermore, stable knockdown of ERbeta resulted in loss of growth contact inhibition. In this work, we report that loss of ERbeta is associated with a decrease of E-cadherin protein levels through different posttranscriptional regulatory mechanisms. Ligand activation of ERalpha induced E-cadherin extracellular shedding and internalization only in the absence of ERbeta, followed by lysosomal degradation. Loss of ERbeta also led to an increase of E-cadherin uptake in a ligand-independent manner through mechanisms that required caveolae formation. Proteasome activity was necessary for both mechanisms to operate. Increased E-cadherin internalization correlated with the up-regulation of beta-catenin transcriptional activity and impaired morphogenesis on Engelbreth-Holm-Swarm matrix. Taken together, these results emphasize the role of epithelial ERbeta in maintaining cell adhesion and a differentiated phenotype and highlight the potential importance of ERbeta for the design of specific agonists for use in breast cancer therapy.

Species difference exists in the effects of 1alpha,25(OH)(2)D(3) and its analogue 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD) on osteoblastic cells.

The direct effect of 1alpha,25(OH)(2)D(3) on osteoblasts remains unclear. In this study, we evaluated the in vitro effects of 1alpha,25(OH)(2)D(3) and its analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD), on osteoblasts from three different species, i.e. bone marrow stromal cells from the Sprague-Dawley (SD) rat, from the C57BL/6 mouse, as well as human osteoblast NHOst cells and human osteosarcoma derived MG-63 cells. We found that in rat cells, both compounds increased cell proliferation, inhibited cell apoptosis and increased alkaline phosphatase (ALP) activity. In mouse cells, however, both compounds initiated cell apoptosis and inhibited ALP activity. In human cells, although cell proliferation was inhibited by both compounds, cell apoptosis was inhibited and ALP activity was enhanced. In each species, 2MD was much more potent than 1alpha,25(OH)(2)D(3). To summarize, species differences should be taken into account in studies of vitamin D effects. However, in all tested species - rat, mouse and human - 2MD is considerably more potent in its effects on osteoblastic cells in vitro than 1alpha,25(OH)(2)D(3).