Ko-En Huang

From transforming growth factor-β signaling to androgen action: Identification of Smad3 as an androgen receptor coregulator in prostate cancer cells

Although transforming growth factor-β (TGF-β) has been identified to mainly inhibit cell growth, the correlation of elevated TGF-β with increasing serum prostate-specific antigen (PSA) levels in metastatic stages of prostate cancer has also been well documented. The molecular mechanism for these two contrasting effects of TGF-β, however, remains unclear. Here we report that Smad3, a downstream mediator of the TGF-β signaling pathway, functions as a coregulator to enhance androgen receptor (AR)-mediated transactivation. Compared with the wild-type AR, Smad3 acts as a strong coregulator in the presence of 1 nM 5α-dihydrotestosterone, 10 nM 17β-estradiol, or 1 μM hydroxyflutamide for the LNCaP mutant AR (mtAR T877A), found in many prostate tumor patients. We further showed that endogenous PSA expression in LNCaP cells can be induced by 5α-dihydrotestosterone, and the addition of the Smad3 further induces PSA expression. Together, our findings establish Smad3 as an important coregulator for the androgen-signaling pathway and provide a possible explanation for the positive role of TGF-β in androgen-promoted prostate cancer growth.

Activin A Enhances Prostate Cancer Cell Migration Through Activation of Androgen Receptor and Is Overexpressed in Metastatic Prostate Cancer

Bone metastasis is the major cause of mortality associated with prostate cancer. Whereas activin A is known to inhibit prostate cancer cell growth and promote apoptosis, the correlation of elevated activin A with increasing serum prostate‐specific antigen (PSA) levels in bone metastatic stages of prostate cancer is well documented. The molecular mechanisms explaining these paradoxical effects of activin A and how activin A influences the progression of prostate cancer with bone metastasis remain unclear. By comparing expression profiles of primary prostate cancer biopsies, with and without bone metastasis, we discovered that the expression of activin A is increased in cases with bone metastatic propensity and correlates with increased androgen receptor (AR), PSA expression, and Gleason scores. Activin A promotes migration of prostate cancer cells to osteoblasts, elevates the AR gene transcription through Smads through binding to AR promoter, and induces nuclear translocation of AR to interact with Smad3. Knockdown of Smad3 by siRNA decreases activin A–promoted AR expression and cancer cell migration. Overexpression of AR reversed Smad3‐siRNA suppression on activin A–mediated cell migration to osteoblasts. These data suggest that activation of the AR through Smads is required for activin A–promoted prostate cancer cell migration to bone matrix, thereby promoting the bone metastatic phenotype, and the activin A–Smad–AR axis may be considered a therapeutic target in bone metastatic diseases.

Comparison of transdermal and oral estrogen-progestin replacement therapy: effects on cardiovascular risk factors.

ObjectiveTo determine the effects of oral and transdermal hormone replacement therapy on lipid profile and hemostatic factors in postmenopausal women. DesignTwenty subjects were treated with oral E2 valerate (2 mg) combined with cyproterone acetate (1 mg) (group I) and 21 with transdermal E2 (1.5 mg) plus oral medroxyprogesterone acetate (5 mg) (group II). The effects on lipid profile and hemostatic parameters were evaluated at baseline and after 3, 6, and 12 months of treatment. ResultsGroup I showed a stronger increase of high-density lipoprotein (HDL) cholesterol levels (2–8%) and stronger reduction of atherogenic indices (total cholesterol/HDL cholesterol and low-density lipoprotein/HDL cholesterol) than group II. Group II showed a more pronounced reduction of triglyceride (21–31%) and factor VII (6–10%) levels than group I. Both groups showed reduced concentrations of total cholesterol, low-density lipoprotein cholesterol, tissue plasminogen activator, plasminogen activator inhibitor-1, antithrombin III, and protein S, whereas protein C was increased after 12 months of treatment. ConclusionsThe cardioprotective effects of hormone replacement therapy are demonstrated by favorable effects on lipid profile and fibrinolytic activity. Oral hormone replacement therapy showed a more prominent effect on lipoprotein metabolism than did transdermal administration, but transdermal medication had a stronger effect on triglyceride and coagulation factors. However, it needs to be considered that there is an increased risk of venous thrombotic events in the first year of treatment.

Tumor Suppressor PAX6 Functions as Androgen Receptor Co-Repressor to Inhibit Prostate Cancer Growth

PAX6, a transcription factor, has currently been suggested to function as a tumor suppressor in glioblastoma and to act as an early differentiation marker for neuroendocrine cells. The androgen receptor (AR) plays a pivotal role in prostate cancer development and progression due to its transcriptional activity in regulating genes involved in cell growth, differentiation, and apoptosis. To determine the role of PAX6 in prostate cancer, we investigated whether PAX6 interacts with AR to affect prostate cancer development.

Cryptotanshinone down-regulates androgen receptor signaling by modulating lysine-specific demethylase 1 function

Development and progression of prostate cancer are intimately associated with androgen receptor (AR) signaling. The emergence of hormone‐refractory prostate cancer and consequent failure of conventional androgen deprivation therapies make it necessary to bypass hormonal resistance by targeting the same signaling pathway at new intervention points. In our study, we showed that cryptotanshinone inhibited the growth of AR‐positive prostate cancer cells, suggesting that cryptotanshinone affected AR function. Cryptotanshinone also profoundly inhibited the transcriptional activity of AR and suppressed the expression of several AR‐target genes at the mRNA and the protein levels. At the molecular level, cryptotanshinone disrupted the interaction between AR and lysine‐specific demethylase 1 (LSD1), and inhibited the complex of AR and LSD1 to the promoter of AR target genes without affecting the protein degradation and translocation of AR. Cryptotanshinone increased the mono‐methyl and di‐methylation of Histone H3 lysine 9 (H3K9), a repressive histone marker which is demethylated and activated by LSD1. These data suggest that cryptotanshinone functions via inhibition of LSD1, a protein that promotes AR‐dependent transcriptional activity via derepression of H3K9. In summary, we describe a novel mechanism whereby cryptotanshinone down‐regulates AR signaling via functional inhibition of LSD1‐mediated demethylation of H3K9 and represses the transcriptional activity of AR. Our data suggest that cryptotanshinone can be developed as a potential therapeutic agent for prostate cancer.

Roles of testicular orphan nuclear receptors 2 and 4 in early embryonic development and embryonic stem cells.

The testicular orphan nuclear receptors (TRs) 2 and 4 act as either transcriptional activators or regulatory proteins of other nuclear receptor superfamily members. With no identified cognate ligands, their physiological roles remain unclear. Here we showed the phenotypes of TR2(-/-):TR4(-/-) mutant embryos, which reveal that the loss of TR2 and TR4 causes early embryonic lethality and increased cell death. We also found that TR2 and TR4 are expressed in blastocysts and embryonic stem (ES) cells, and can act as transcriptional activators in ES cells. The results on further investigating the roles of TR2 and TR4 in ES cells showed that TR2 and TR4 were differentially expressed when ES cells were induced into different specialized cell types, and their expression is regulated by retinoic acid. Knocking down TR2 and TR4 mRNAs decreased the expression of Oct-3/4 and Nanog genes. Mechanism dissection suggests that TR2 and TR4 may affect the Oct-3/4 gene by binding to a direct repeat-1 element located in its promoter region, which is influenced by retinoic acid. Together, our findings highlight possible roles for TR2 and TR4 in early embryonic development by regulating key genes involved in stem cell self-renewal, commitment, and differentiation.

Effect of estrogen and 1α,25(OH)2- vitamin D3 on the activity and growth of human primary osteoblast-like cells in vitro

Abstract Objective: To evaluate effects of 17β-E 2 and 1α,25(OH) 2 -vitamin D 3 on human osteoblast-like (hOB) cells. Design: Controlled, experimental study. Setting: University hospital. Patient(s): hOB cell cultures were prepared from the upper femur of postmenopausal patients undergoing bipolar endoprosthesis arthroplasty for a fractured femoral neck. Intervention(s): hOB cells were subcultured with either 17β-E 2 or 1α,25(OH) 2 -vitamin D 3 , or both. Main Outcome Measure(s): Cell proliferation and activity of alkaline phosphatase, osteocalcin, and interleukin-6. Result(s): 17β-E 2 significantly reduced interleukin-6 and osteocalcin to 34% and 60% of control value but induced alkaline phosphatase and cell proliferation to 183% and 150% of control value. 1α,25(OH) 2 -vitamin D 3 significantly decreased cell proliferation to 88% of that of control group, but 1α,25(OH) 2 -vitamin D 3 plus 17β-E 2 showed no difference from the control group. Alkaline phosphatase and osteocalcin were significantly increased by 1α,25(OH) 2 -vitamin D 3 alone or combined with 17β-E 2 , to 169% and 198% and to 144% and 144% of control value, respectively. 1α,25(OH) 2 -vitamin D 3 , with or without 17β-E 2 , decreased interleukin-6 levels to 27% and 38% of control group, respectively. Conclusion(s): 17β-E 2 and 1α,25(OH) 2 -vitamin D 3 have effects on osteoblasts. The prevention of osteoporosis by estrogen may be related not only to direct effects on osteoblastic activity and proliferation but also to indirect effects on osteoclasts by the decrease of interleukin-6 secretion.

The reduced trabecular bone mass of adult ARKO male mice results from the decreased osteogenic differentiation of bone marrow stroma cells

Male mice with androgen receptor knock-out (ARKO) show significant bone loss at a young age. However, the lasting effect of AR inactivation on bone in aging male mice remains unclear. We designed this study to evaluate the effect of AR on bone quality in aging male mice and to find the possible causes of AR inactivation contributing to the bone loss. The mice were grouped according to their ages and AR status and their trabecular bones were examined by micro-CT analysis at 6, 12, 18, and 30 weeks old. We found that bone mass consistently decreased and the bone microarchitectures continuously deteriorated in male ARKO mice at designated time points. To determine the cause of the bone loss in ARKO mice, we further examined the role of AR in bone cell fate decision and differentiation and we conducted experiments on bone marrow stromal cells (BMSC) obtained from wild type (WT) and AR knockout (KO) mice. We found that ARKO mice had higher numbers of colony formation unit-fibroblast (CFU-F), and CD44 and CD34 positive cells in bone marrow than WT mice. Our Q-RT-PCR results showed lower expression of genes linked to osteogenesis in BMSCs isolated from ARKO mice. In conclusion, AR nullification disrupted bone microarchitecture and caused trabecular bone mass loss in male ARKO mice. And the fate of BMSCs was impacted by the loss of AR. Therefore, these findings suggest that AR may accelerate the use of progenitor cells and direct them into osteogenic differentiation to affect bone metabolism.

Altered TNSALP Expression and Phosphate Regulation Contribute to Reduced Mineralization in Mice Lacking Androgen Receptor

ABSTRACT While androgen receptor (AR)-deficient mice developed osteopenia in endochondral bones due to the high bone turnover with increased bone resorption by osteoclasts, little is known about the mechanism of intramembranous bone loss contributed by AR in osteoblasts. Here, we discovered a dramatic decrease in the area of calcification, new bone, and the number of osteocytes in calvaria from AR-deficient mice related to a reduction in mineralization caused, in part, by the diminished activity of AR-deficient osteoblasts. Enforced AR expression in differentiated osteoblasts boosts mineralization while knockdown of AR expression prevents androgen-induced mineralization. We identified the tissue-nonspecific alkaline phosphatase (TNSALP) and several members of small integrin binding ligand N-linked glycoprotein (SIBLING) gene family as androgen target genes required for AR-mediated bone formation. We show that inorganic phosphate (Pi) levels and TNSALP activity increased in response to androgen/AR and Pi signals increase the expression and translocation of AR. The ectopic expression of TNSALP or Pi partially rescued the bone loss due to AR deficiency. Thus, androgen/AR signaling plays an essential role in bone formation by coordinating the expression of genes associated with phosphate regulation.

Effects of standardized phytoestrogen on Taiwanese menopausal women

OBJECTIVE To investigate the effects of standardized soy extract on climacteric symptoms, lipid profiles, bone markers, and serum isoflavone concentration in healthy Taiwanese postmenopausal women. MATERIALS AND METHODS A multicenter, open-labeled, randomized, prospective, comparative study design was used. A total of 130 outpatients who had undergone natural menopause were randomly administered either 70 mg or 35 mg soy extract daily for 24 weeks. RESULTS The evidence suggests that the soy extract treatment that was administered to both groups for 1 month could help reduce climacteric scores (reductions of 19.66% [p<0.01] and 18.85% [p<0.01] in the 35 mg and 70 mg groups compared with baseline, respectively), and the efficacy was more potent after 6 months of treatment. Soy isoflavone significantly reduced the total cholesterol (reductions of 4.50% [p<0.01] and 3.06% [p<0.05] in the 35 mg and 70 mg groups, respectively) and low density lipoprotein cholesterol levels (reductions of 4.67% [p<0.05] and 5.09% [p<0.05] in the 35 mg and 70 mg groups, respectively) in patients with total cholesterol > 200 mg/dL after 6 months of treatment. In patients with high bone turnover (urinary deoxypyridinoline/creatinine > 7.4 nM/mM), soy extract treatment reduced the deoxypyridinoline/creatinine level by 10.53% (p<0.05) and 11.58% (p<0.05) in the 35 mg and 70 mg groups, respectively. Serum levels of isoflavone increased in both groups after 6 months of treatment. CONCLUSION Soy extract is highly efficacious at relieving menopausal symptoms and demonstrates a positive effect on the cardiovascular system and skeleton.