Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes.

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells.

Estrogen receptor α mediates proliferation of osteoblastic cells stimulated by estrogen and mechanical strain, but their acute down-regulation of the Wnt antagonist Sost is mediated by estrogen receptor β.

Mechanical strain and estrogens both stimulate osteoblast proliferation through estrogen receptor (ER)-mediated effects, and both down-regulate the Wnt antagonist Sost/sclerostin. Here, we investigate the differential effects of ERα and -β in these processes in mouse long bone-derived osteoblastic cells and human Saos-2 cells. Recruitment to the cell cycle following strain or 17β-estradiol occurs within 30 min, as determined by Ki-67 staining, and is prevented by the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride.

A specific role for phosphoinositide 3-kinase and AKT in osteoblasts?

The phosphoinositide 3-kinase and AKT (protein kinase B) signaling pathway (PI3K/AKT) plays a central role in the control of cell survival, growth, and proliferation throughout the body. With regard to bone, and particularly in osteoblasts, there is an increasing amount of evidence that the many signaling molecules exert some of their bone-specific effects in part via selectively activating some of the generic effects of the PI3K/AKT pathway in osteoblasts. There is further data demonstrating that PI3K/AKT has the capacity to specifically cross-talk with other signaling pathways and transcriptional networks controlling bone cells' development in order to fine-tune the osteoblast phenotype.

Lrp5 is not required for the proliferative response of osteoblasts to strain but regulates proliferation and apoptosis in a cell autonomous manner.

Although Lrp5 is known to be an important contributor to the mechanisms regulating bone mass, its precise role remains unclear. The aim of this study was to establish whether mutations in Lrp5 are associated with differences in the growth and/or apoptosis of osteoblast-like cells and their proliferative response to mechanical strain in vitro. Primary osteoblast-like cells were derived from cortical bone of adult mice lacking functional Lrp5 (Lrp5(-/-)), those heterozygous for the human G171V High Bone Mass (HBM) mutation (LRP5(G171V)) and their WT littermates (WT(Lrp5), WT(HBM)).

Loading-related regulation of transcription factor EGR2/Krox-20 in bone cells is ERK1/2 protein-mediated and prostaglandin, Wnt signaling pathway-, and insulin-like growth factor-I axis-dependent.

Of the 1,328 genes revealed by microarray to be differentially regulated by disuse, or at 8 h following a single short period of osteogenic loading of the mouse tibia, analysis by predicting associated transcription factors from annotated affinities revealed the transcription factor EGR2/Krox-20 as being more closely associated with more pathways and functions than any other. Real time quantitative PCR confirmed up-regulation of Egr2 mRNA expression by loading of the tibia in vivo. In vitro studies where strain was applied to primary cultures of mouse tibia-derived osteoblastic cells and the osteoblast UMR106 cell line also showed up-regulation of Egr2 mRNA expression.

Sost down-regulation by mechanical strain in human osteoblastic cells involves PGE2 signaling via EP4.

Sclerostin is a potent inhibitor of bone formation which is down-regulated by mechanical loading. To investigate the mechanisms involved we subjected Saos2 human osteoblastic cells to short periods of dynamic strain and used quantitative reverse transcriptase polymerase chain reaction to compare their responses to unstrained controls. Strain-induced Sost down-regulation was recapitulated by cyclo-oxygenase-2-mediated PGE2, acting through the EP4 receptor, whereas strain-related up-regulation of osteocalcin was mediated by the EP2 receptor.

Role of endocrine and paracrine factors in the adaptation of bone to mechanical loading.

There appears to be no unique mechanically sensitive pathway by which changes in bone loading regulate bone mass and architecture to ensure adequate structural strength. Rather, strain-derived changes in bone cells activate a number of nonspecific strain-sensitive pathways (including calcium fluxes, prostanoids, nitric oxide, extracellular signal-regulated kinase, and sclerostin), the activities of which are modified by a number of factors (including estrogen receptors) for which this contribution is subsidiary to other purposes. The strain-sensitive pathways modified by these factors interact with a number of other pathways, some of which appear to have specific osteoregulatory potential (eg, the parathyroid hormone pathway), whereas others such as the Wnt pathway appear to be associated primarily with the response mechanisms of proliferation, differentiation, and apoptosis.

Constitutively nuclear FOXO3a localization predicts poor survival and promotes Akt phosphorylation in breast cancer.

Background: The PI3K-Akt signal pathway plays a key role in tumorigenesis and the development of drug-resistance. Cytotoxic chemotherapy resistance is linked to limited therapeutic options and poor prognosis.

Methodology/principal Findings: Examination of FOXO3a and phosphorylated-Akt (P-Akt) expression in breast cancer tissue microarrays showed nuclear FOXO3a was associated with lymph node positivity (p = 0.

Mechano-transduction in osteoblastic cells involves strain-regulated estrogen receptor alpha-mediated control of insulin-like growth factor (IGF) I receptor sensitivity to Ambient IGF, leading to phosphatidylinositol 3-kinase/AKT-dependent Wnt/LRP5 receptor-independent activation of beta-catenin signaling.

The capacity of bones to adjust their mass and architecture to withstand the loads of everyday activity derives from the ability of their resident cells to respond appropriately to the strains engendered. To elucidate the mechanisms of strain responsiveness in bone cells, we investigated in vitro the responses of primary mouse osteoblasts and UMR-106 osteoblast-like cells to a single period of dynamic strain. This stimulates a cascade of events, including activation of insulin-like growth factor I receptor (IGF-IR), phosphatidylinositol 3-kinase-mediated phosphorylation of AKT, inhibition of GSK-3beta, increased activation of beta-catenin, and associated lymphoid-enhancing factor/T cell factor-mediated transcription.

Loading-related regulation of gene expression in bone in the contexts of estrogen deficiency, lack of estrogen receptor alpha and disuse.

Loading-related changes in gene expression in resident cells in the tibia of female mice in the contexts of normality (WT), estrogen deficiency (WT-OVX), absence of estrogen receptor alpha (ERalpha(-/-)) and disuse due to sciatic neurectomy (WT-SN) were established by microarray. Total RNA was extracted from loaded and contra-lateral non-loaded tibiae at selected time points after a single, short period of dynamic loading sufficient to engender an osteogenic response. There were marked changes in the expression of many genes according to context as well as in response to loading within those contexts.

The mouse fibula as a suitable bone for the study of functional adaptation to mechanical loading.

Bones' functionally adaptive responses to mechanical loading can usefully be studied in the tibia by the application of loads between the knee and ankle in normal and genetically modified mice. Such loading also deforms the fibula. Our present study was designed to ascertain whether the fibula adapts to loading in a similar way to the tibia and could thus provide an additional bone in which to study functional adaptation.

Mechanical loading enhances the anabolic effects of intermittent parathyroid hormone (1-34) on trabecular and cortical bone in mice.

The separate and combined effects of intermittent parathyroid hormone (iPTH) (1-34) and mechanical loading were assessed at trabecular and cortical sites of mouse long bones. Female C57BL/6 mice from 13 to 19 weeks of age were given daily injections of vehicle or PTH (1-34) at low (20 microg/kg/day), medium (40 microg/kg/day) or high (80 microg/kg/day) dose. For three alternate days per week during the last two weeks of this treatment, the tibiae and ulnae on one side were subjected to a single period of non-invasive, dynamic axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle).

The transcription factor FOXO3a is a crucial cellular target of gefitinib (Iressa) in breast cancer cells.

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) that causes growth delay in cancer cell lines and human tumor xenografts expressing high levels of EGFR. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-EGFR therapies and provide information for key targets for therapeutic intervention. In this study, we investigated the role of FOXO3a in gefitinib action and resistance.

Wnt/beta-catenin signaling is a component of osteoblastic bone cell early responses to load-bearing and requires estrogen receptor alpha.

The Wnt/beta-catenin pathway has been implicated in bone cell response to their mechanical environment. This response is the origin of the mechanism by which bone cells adjust bone architecture to maintain bone strength. Osteoporosis is the most widespread failure of this mechanism.

Paclitaxel-induced nuclear translocation of FOXO3a in breast cancer cells is mediated by c-Jun NH2-terminal kinase and Akt.

The microtubule-targeting compound paclitaxel is often used in the treatment of endocrine-resistant or metastatic breast cancer. We have previously shown that apoptosis of breast cancer cells in response to paclitaxel is mediated by induction of FOXO3a expression, a transcription factor downstream of the phosphatidylinositol-3-kinase/Akt signaling pathway. To further investigate its mechanism of action, we treated MCF-7 cells with paclitaxel and showed a dose-dependent increase in nuclear localization of FOXO3a, which coincided with decreased Akt signaling but increased c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) activity.

ICI182,780 induces p21Waf1 gene transcription through releasing histone deacetylase 1 and estrogen receptor alpha from Sp1 sites to induce cell cycle arrest in MCF-7 breast cancer cell line.

We used the estrogen-responsive MCF-7 breast cancer cell line as a relevant model to study the anti-proliferative effects of ICI182,780 and identified the negative cell cycle regulator p21Waf1 as a specific target of ICI182,780. Furthermore, silencing of the p21Waf1 expression by small interfering RNA overcame the G0/G1 cell cycle arrest induced by ICI182,780, suggesting that the induction of p21Waf1 expression has a direct role in mediating the ICI182,780-induced G0/G1 arrest. We further demonstrated that the induction of p21Waf1 by ICI182,780 is mediated at transcriptional and gene promoter levels through the proximal Sp1 sites located near the transcription start site.

Aberrant p53 alters DNA damage checkpoints in response to cisplatin: downregulation of CDK expression and activity.

The p53 tumor suppressor protein is a critical mediator of cell cycle arrest and apoptosis in response to genotoxic stress. Abrogation of p53 function is a major feature of tumor development and may result in a compromised DNA-damage response. In our study, we examined the effect of expressing a human p53 cDNA, encoding a histidine to leucine amino acid substitution at codon 179 (H179L), on the ability of wild-type p53-containing NIH3T3 cells to respond to treatment with the chemotherapeutic cisplatin.

The expression of ER beta cx in human breast cancer and the relationship to endocrine therapy and survival.

Purpose: Estrogen receptor (ER) alpha-positive breast cancer is often treated with endocrine therapy using either antiestrogens or aromatase inhibitors. However, 30% of patients who receive endocrine therapy will derive no benefit from such treatments and may indeed suffer adverse effects. Currently, there are no ways to predict response to such treatments.

Accelerated cell cycle progression in osteoblasts overexpressing the c-fos proto-oncogene: induction of cyclin A and enhanced CDK2 activity.

Transgenic mice overexpressing the c-Fos oncoprotein develop osteosarcomas that are associated with deregulated expression of cell cycle genes. Here we have generated osteoblast cell lines expressing c-fos under the control of a tetracycline-regulatable promoter to investigate the role of c-Fos in osteoblast cell cycle control in vitro. Three stable subclones, AT9.

Expression of estrogen receptor beta isoforms in normal breast epithelial cells and breast cancer: regulation by methylation.

Two novel estrogen receptor beta (ERbeta) mRNA isoforms that diverge in their 5'-untranslated regions, ERbeta mRNA (0K-1) and ERbeta mRNA (0N-1), have recently been identified. This indicates that transcription of the human ERbeta gene occurs from at least two different promoters, named promoter 0K and promoter 0N. The aim of this study was to investigate the expression of ERbeta isoforms in primary cultures of normal breast epithelial cells, a panel of breast cancer cell lines and in normal breast and breast cancer tissues; and to examine whether methylation of the two ERbeta promoters is involved in regulation of ERbeta gene expression.

FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines.

Paclitaxel is used to treat breast cancers, but the mechanisms by which it induces apoptosis are poorly understood. Consequently, we have studied the role of the FoxO transcription factors in determining cellular response to paclitaxel. Western blotting revealed that in a panel of nine breast cancer cell lines expression of FoxO1a and FoxO3a correlated with the expression of the pro-apoptotic FoxO target Bim, which was associated with paclitaxel-induced apoptosis.