Anne Gingery

Phosphatidylinositol 3‐kinase coordinately activates the MEK/ERK and AKT/NFκB pathways to maintain osteoclast survival

We have examined highly purified osteoclasts that were generated in vitro from murine co‐culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFκB survival pathways. Many mature marrow‐derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M‐CSF had no impact on their survival pattern. In addition, spleen‐derived osteoclasts generated with RANKL and M‐CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFκB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow‐derived osteoclasts, marrow‐derived osteoclasts co‐cultured with stromal cells, and spleen‐derived osteoclasts maintained with RANKL and M‐CSF. These data support that both the MEK/ERK and AKT/NFκB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co‐cultured marrow‐derived osteoclasts and spleen‐derived osteoclasts maintained with RANKL and M‐CSF. Interestingly, in marrow‐derived co‐cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFκB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival. J. Cell. Biochem. 89: 165–179, 2003.

TGF-beta coordinately activates TAK1/MEK/AKT/NFkB and SMAD pathways to promote osteoclast survival.

To better understand the roles of TGF-beta in bone metabolism, we investigated osteoclast survival in response TGF-beta and found that TGF-beta inhibited apoptosis. We examined the receptors involved in promotion of osteoclast survival and found that the canonical TGF-beta receptor complex is involved in the survival response. The upstream MEK kinase TAK1 was rapidly activated following TGF-beta treatment. Since osteoclast survival involves MEK, AKT, and NFkappaB activation, we examined TGF-beta effects on activation of these pathways and observed rapid phosphorylation of MEK, AKT, IKK, IkappaB, and NFkappaB. The timing of activation coincided with SMAD activation and dominant negative SMAD expression did not inhibit NFkappaB activation, indicating that kinase pathway activation is independent of SMAD signaling. Inhibition of TAK1, MEK, AKT, NIK, IKK, or NFkappaB repressed TGF-beta-mediated osteoclast survival. Adenoviral-mediated TAK1 or MEK inhibition eliminated TGF-beta-mediated kinase pathway activation and constitutively active AKT expression overcame apoptosis induction following MEK inhibition. TAK1/MEK activation induces pro-survival BclX(L) expression and TAK1/MEK and SMAD pathway activation induces pro-survival Mcl-1 expression. These data show that TGF-beta-induced NFkappaB activation is through TAK1/MEK-mediated AKT activation, which is essential for TGF-beta to support of osteoclast survival.

Endoxifen’s Molecular Mechanisms of Action Are Concentration Dependent and Different than That of Other Anti-Estrogens

Endoxifen, a cytochrome P450 mediated tamoxifen metabolite, is being developed as a drug for the treatment of estrogen receptor (ER) positive breast cancer. Endoxifen is known to be a potent anti-estrogen and its mechanisms of action are still being elucidated. Here, we demonstrate that endoxifen-mediated recruitment of ERα to known target genes differs from that of 4-hydroxy-tamoxifen (4HT) and ICI-182,780 (ICI). Global gene expression profiling of MCF7 cells revealed substantial differences in the transcriptome following treatment with 4HT, endoxifen and ICI, both in the presence and absence of estrogen. Alterations in endoxifen concentrations also dramatically altered the gene expression profiles of MCF7 cells, even in the presence of clinically relevant concentrations of tamoxifen and its metabolites, 4HT and N-desmethyl-tamoxifen (NDT). Pathway analysis of differentially regulated genes revealed substantial differences related to endoxifen concentrations including significant induction of cell cycle arrest and markers of apoptosis following treatment with high, but not low, concentrations of endoxifen. Taken together, these data demonstrate that endoxifen’s mechanism of action is different from that of 4HT and ICI and provide mechanistic insight into the potential importance of endoxifen in the suppression of breast cancer growth and progression.

Exercise Training Attenuates Placental Ischemia-Induced Hypertension and Angiogenic Imbalance in the Rat

An imbalance between proangiogenic (vascular endothelial growth factor) and antiangiogenic (soluble fms-like tyrosine kinase 1) factors plays an important role in hypertension associated with reduced uteroplacental perfusion (RUPP). Exercise has been shown to stimulate proangiogenic factors, such as vascular endothelial growth factor, in both the pregnant and nonpregnant state; thus, we hypothesized that exercise training would attenuate both angiogenic imbalance and hypertension attributed to RUPP. Four groups of animals were studied, RUPP and normal pregnant controls and normal pregnant and RUPP+exercise training. Exercise training attenuated RUPP-induced hypertension (P<0.05), decreased soluble fms-like tyrosine kinase 1 (P<0.05), increased VEGF (P<0.05), and elevated the soluble fms-like tyrosine kinase 1:vascular endothelial growth factor ratio. The positive effects of exercise on angiogenic balance in the RUPP rats were confirmed by restoration (P<0.05) of the RUPP-induced decrease in endothelial tube formation in human umbilical vascular endothelial cells treated with serum from each of the experimental groups. Placental prolyl hydroxylase 1 was increased (P<0.05) in RUPP+exercise training rats. Decreased trolox equivalent antioxidant capacity in the placenta, amniotic fluid, and kidney of the RUPP rats was reversed by exercise. RUPP-induced increase in renal thiobarbituric acid reactive species was attenuated by exercise. The present data show that exercise training before and during pregnancy attenuates placental ischemia-induced hypertension, angiogenic imbalance, and oxidative stress in the RUPP rat and reveals that increased prolyl hydroxylase 1 is associated with decreased soluble fms-like tyrosine kinase 1, thus revealing several potential pathways for exercise training to mitigate the effects of placental ischemia-induced hypertension. Lastly, the present study demonstrates that exercise training may be a useful approach to attenuate the development of placental ischemia-induced hypertension during pregnancy.

Chronic placental ischemia alters amniotic fluid milieu and results in impaired glucose tolerance, insulin resistance and hyperleptinemia in young rats

Although small size at birth is associated with hypertension and associated co-morbidities such as insulin resistance and type II diabetes mellitus, many of the animal models employed to simulate this phenomenon do not closely mimic the ontogeny of growth restriction observed clinically. While intrauterine growth restriction (IUGR) is often detected near mid-pregnancy in women and persists until term, most rodent models of IUGR employ ligation of uterine arteries for a brief period during late gestation (days 19–21 of pregnancy). We hypothesized that IUGR associated with chronic reduction in uteroplacental perfusion (RUPP) and placental ischemia during the third trimester of pregnancy in the rat alters the amniotic fluid (AF) environment and results in hypertensive offspring presenting with metabolic abnormalities such as glucose intolerance and insulin resistance. Insulin-like growth factor-1 (IGF-1), IGF-2, Na+ concentration and oxidative stress in the AF were increased, while K+ concentration was decreased in the RUPP compared with normal pregnant (NP) fetuses. RUPP-offspring (RUPP-O) were smaller (6.1 ± 0.2 versus 6.7 ± 0.2 g; P < 0.05) at birth compared with NP-offspring (NP-O) groups. At nine weeks of age, mean arterial pressure (121 ± 3 versus 107 ± 5 mmHg; P < 0.05), fasting insulin (0.71 ± 0.014 versus 0.30 ± 0.08 ng/mL; P < 0.05), glucose (4.4 ± 0.2 versus 3.1 ± 0.3 mmol/L; P < 0.05), leptin (3.8 ± 0.5 versus 2.3 ± 0.3 ng/mL; P < 0.05) and the homeostasis model assessment of insulin resistance index was greater (2.9 ± 0.6 versus 1.0 ± 0.3; P < 0.05) in the RUPP-O compared with the NP-O rats. These data indicate that chronic placental ischemia results in numerous alterations to the fetal environment that contributes to the development of impaired glucose metabolism, insulin resistance and hyperleptinemia in young offspring.

ERβ1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer

BackgroundThe role and clinical value of ERβ1 expression is controversial and recent data demonstrates that many ERβ antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERβ1 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.MethodsNuclear and cytoplasmic expression patterns of ERβ1 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 ERβ1, we generated multiple ERβ1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERβ-specific agonist exposure.ResultsNuclear ERβ1 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, ERβ1 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, ERβ-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective.ConclusionsUsing a validated antibody, we have confirmed that nuclear ERβ1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERβ appears to be a novel therapeutic target. However, the efficacy of SERMs and ERβ-specific agonists differ as a function of ERα expression.

Placental and vascular adaptations to exercise training before and during pregnancy in the rat

Although exercise during pregnancy is generally recommended and thought to be beneficial to mother and fetus, the nature of the adaptations to exercise during pregnancy and how they may be beneficial remain poorly understood. Recent studies suggest that exercise may stimulate expression of several cytoprotective and pro-angiogenic molecules such as heat shock proteins (HSP) and vascular endothelial growth factors (VEGF). We hypothesized that exercise training during pregnancy improves angiogenic balance, increases HSP expression, and improves endothelial function. Female rats were given access to an exercise wheel for 6 wk before and during pregnancy. On day 19 of pregnancy tissues were collected and snap frozen for later analysis. Western blots were performed in skeletal muscle and placenta. HSP 27 (3.7 ± 0.36 vs. 2.2 ± 0.38; P < 0.05), HSP 60 (2.2 ± 0.73 vs. 0.49 ± 0.08; P < 0.05), and HSP 90 (0.33 ± 0.09 vs. 0.11 ± 0.02; P < 0.05) were increased in the placentas of exercise-trained rats compared with sedentary controls. In addition, exercise training increased (P < 0.05) plasma free VEGF and augmented (P < 0.05) endothelium-dependent vascular relaxation compared with nonexercise control rats. The present data indicates chronic exercise training stimulates HSP expression in the placenta and that regular exercise training increases circulating VEGF in pregnant but not in nonpregnant rats. Although the present findings suggest that exercise before and during pregnancy may promote the expression of molecules that could attenuate placental and vascular dysfunction in complicated pregnancies, further studies are needed to determine the safety and effectiveness of exercise training as a therapeutic modality in pregnancy.

Transforming growth factor-β (TGF-β) expression is increased in the subsynovial connective tissues of patients with idiopathic carpal tunnel syndrome.

Non‐inflammatory fibrosis of the subsynovial connective tissue (SSCT) is a hallmark of carpal tunnel syndrome (CTS). The etiology of this finding and its relationship to the development of CTS remain poorly understood. Recent studies have found that transforming growth factor‐β (TGF‐β) plays a central role in fibrosis. The purpose of this study was to investigate the expression of TGF‐β and connective tissue growth factor (CTGF), a downstream mediator of TGF‐β, in the pathogenesis of CTS. We compared SSCT specimens from 26 idiopathic CTS patients with specimens from 10 human cadaver controls with no previous diagnosis of CTS. Immunohistochemistry was performed to determine levels TGF‐β1, CTGF, collagen 1(Col1) and collagen 3 (Col3) expression. TGF‐β1 (p < 0.01), CTGF (p < 0.01), and Col3 (p < 0.01) were increased in SSCT of CTS patients compared with control tissue. In addition, a strong positive correlation was found between TGF‐β1 and CTGF, (R2 = 0.80, p < 0.01) and a moderate positive correlation between Col3 and TGF‐β1 (R2 = 0.49, p < 0.01). These finding suggest that there is an increased expression of TGF‐β and CTGF, a TGF‐β regulated protein, and that this TGF‐β activation may be responsible for SSCT fibrosis in CTS patients.

Timing of ischemic insult alters fetal growth trajectory, maternal angiogenic balance, and markers of renal oxidative stress in the pregnant rat

Increased uterine artery resistance and angiogenic imbalance characterized by increased soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased free vascular endothelial growth factor (VEGF) are often associated with placental insufficiency and preeclampsia but not synonymous with hypertension. We hypothesized chronic reductions in utero-placental perfusion (RUPP) for 5 days (d) during either mid- (d12-d17) or late (d14-d19) gestation would have disparate effects on plasma sFlt-1 and VEGF levels and blood pressure. Five days of chronic RUPP was achieved by placement of silver clips on the abdominal aorta and ovarian arteries on either gestational d12 or d14. Arterial pressure was increased (P < 0.05) in RUPP vs. normal pregnant (NP) in both d17 (10%) and d19 (25%) groups, respectively. Circulating free VEGF was decreased (P < 0.05) and sFlt-1:VEGF ratio increased (P < 0.05) after 5 days of RUPP ending on d19 but not d17 compared with NP controls. Angiogenic imbalance, measured by an endothelial tube formation assay, was present in the d19 RUPP but not the d17 RUPP compared with age-matched NP rats. Five days of RUPP from days 14 to 19 decreased fetal and placental weights 10% (P < 0.01) compared with d19 NP controls. After 5 days of RUPP, from days 12 to 17 of pregnancy, fetal weights were 21% lighter (P < 0.01) compared with d17 NP controls, but placental weight was unchanged. These findings suggest that the timing during which placental insufficiency occurs may play an important role in determining the extent of alterations in angiogenic balance, fetal growth restriction, and the severity of placental ischemia-induced hypertension.

TGF-β signaling regulates fibrotic expression and activity in carpal tunnel syndrome.

Fibrosis of the subsynovial connective tissue (SSCT) is a predominant feature of carpal tunnel syndrome (CTS). While the nature of CTS has been extensively studied, little is known about the etiology of this disease. We investigated SSCT tissue from patients with CTS and control subjects using fibrosis arrays and cell culture analysis. Twofold changes in fibrotic gene expression were found in multiple genes from patient SSCT using fibrosis arrays. This data was confirmed via qRT‐PCR on a subset of genes; collagen I (Col1), collagen III (Col3), connective tissue growth factor (CTGF), transforming growth factor β (TGF‐β), and SMAD3 (P < 0.05) which significantly corroborate the fold changes found in the fibrosis arrays. To further explore the nature of SSCT fibrosis, cells were isolated from patient and control tissue. Col1, Col3, TGF‐β, and SMAD3 were highly expressed in patient SSCT fibroblasts as compared to control (P < 0.05). Further, fibrotic genes expression was decreased by inhibiting TGF‐β receptor I (TβRI) activity (P < 0.05). TGF‐β second messenger SMAD activity was significantly activated in SSCT fibroblasts from patients and this activation was abrogated by inhibiting TβRI signaling (P < 0.05). These findings suggest that blocking TGF‐β signaling may be an important therapeutic approach to treating the underlying fibrosis of SSCT in CTS patients.