Chapter 15 Chapter 15 Androgens and Skeletal Biology: Basic Mechanisms Copyright © 2013 Elsevier...

Chapter 15Chapter 15

Androgens and Skeletal Biology: Basic Mechanisms

Copyright © 2013 Elsevier Inc. All rights reserved.


FIGURE 15.1 Principle conversions and major enzyme activities involved in androgen synthesis and metabolism. Steroid hormone synthesis involves metabolism of cholesterol, with dehydrogenation of pregnenolone producing progesterone that can serve as a precursor for the other gonadal steroid hormones. DHEA: dehydroepiandrosterone; CYP11A: cytochrome P450 cholesterol side chain cleavage enzyme; CYP17: cytochrome P450 17α hydroxylase/17,20 lyase; 17β-HSD: 17β-hydroxysteroid dehydrogenase; CYP19: aromatase cytochrome P450.

2


FIGURE 15.2 Model for androgen receptor (AR) regulation of gene expression. Binding of androgen promotes high-affinity dimerization, followed by deoxyribonucleic acid (DNA) binding at the androgen response element (ARE) in an androgen-responsive gene promoter. AR can directly contact TFGIIH and TFIIF in the general transcription machinery. Such interactions between the AR and the general transcription machinery, leading to stable assembly, results in recruitment of ribonucleic acid (RNA) polymerase II and a subsequent increase gene transcript. Coactivators may remodel/modify chromatin through histone acetylation activity to open chromatin structure, or act as a bridge to attract transcript factors (TFs) that trafet binding of TATA-binding protein to the TATTA sequence. Conversely, corepressors act through histone deacetylase activity to reduce accessibility of promoter sequences. Phosphorylation of the receptor may result from activation of SRC by growth factors. Smad3 can act as either a coactivator or corepressor, while cyclin D2 is a corepressor of AR transactivation. Downregulation of gene expression can also be AR mediated.

3


FIGURE 15.3 Dichotomous regulation of androgen receptor (AR) messenger ribonucleic acid (mRNA) levels in osteoblast-like and prostatic carcinoma cell lines after exposure to androgen. A Time course of changes in AR mRNA abundance after 5α-dihydrotestosterone (DHT) exposure in human SaoS-2 osteoblastic cells and human lNCaP prostatic carcinoma cells. To determine the effect of androgen exposure on hAR mRNA abundance, confluent cultures of either osteoblast-like cells (SaoS-S) or prostatic carcinoma cells (lNCaP) were treated with 10–8 M DHT for 0, 24, 48, or 72 hours. Total RNA was then isolated and subjected to RNase protection analysis with 50 μg total cellular RNA from SaoS-2 osteoblastic cells and 10 μg total RNA from cultures. B Densitometic analysis of AR mRNA steady-state levels. The AR mRNA to β-actin ratio is expressed as the mean ± standard error of the mean (SEM) compared to the control value from three to five independent assessments [118].

4


FIGURE 15.4 Expression analyses of estrogen receptor (ER)α, ERβ and androgen receptor (AR) during in vitro differentiation in normal rat osteoblastic (roB) cultures. A Normal roB cells were cultured for the indicated number of days during proliferation, matrix maturation, mineralization and postmineralization stages. Total ribonucleic acid (RNA) was isolated and subjected to relative reverse transcriptase polymerase chain reaction (RT-PCR) analysis using primers specific for rat ERα, ERβ, and AR or rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Reverse transcription was conducted with PCR carried out for 40 cycles for the steroid receptors, with parallel reactions performed using GAPDH primers for 25 cycles (all in the linear range). Bands for rat ERα at the predicted 240 base pairs (bp), rat ERβ at 262 bp, rat AR at 276 bp, and GAPDH at 609 bp are shown. B Analyses of ERα, ERβ, and AR mRNA relative abundance. Semi-quantitative analysis of mRNA steady-state expression by relative RT-PCR was performed after scanning the negative image of the photographed gels. Data are expressed in arbitrary units as the ratio of receptor abundance to GAPDH expression, then normalized to expression values at day 4 in preconfluent cultures. Data represent mean ± standard error of the mean (SEM) [121].

5


FIGURE 15.5 Complex effect of androgen on deoxyribonucleic acid (DNA) accumulation in osteoblastic cultures. A kinetics of 5α-dihydrotestosterone (DHT) response in proliferating colAR-MC3T3 cultures measured with colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. Cultures of stably transfected colAR-MC3T3 continuously with 10 -8 M DHT for 2 days led to increased MTT accumulation, but longer treatment for 3 or 5 days resulted in inhibition. Data are mean ± standard error of the mean (SEM) of six to eight dishes with six wells/dish. *p < 0.05; **p < 0.01 (vs. control) [137].

6


FIGURE 15.6 Characterization of osteoblast apoptosis: results of androgen and estrogen treatment during proliferation (day 5) and during differentiation into mature osteoblast/osteocytes cultures (day 29). Apoptosis was assessed at day 5 or day 29 after continuous 5α-dihydrotestosterone (DHT) and estradiol (E 2) treatment (both at 10 –8 M). Apoptosis was induced by etoposide treatment in proliferating cultures and by serum starvation for 48 hr in confluent cultures before isolation, replaced with 0.1% bovine serum albumin (BSA). A Analysis of apoptosis after evaluating deoxyribonucleic acid (DNA) fragmentation by cytoplasmic nucleosome enrichment at day 5. The data are expressed as mean ± standard error of the mean (SEM) (n = 6) from two independent experiments. **p < 0.01, ***p < 0.001 (vs. control). B. Analysis of apoptosis by cytoplasmic nucleosome enrichment analysis at day 29. The data are expressed as mean ± SEM (n = 6) from two independent experiments. **p < 0.01 vs. control (con) [143].

7


FIGURE 15.7 Von kossa silver staining of mineralized bone nodules over alkaline phosphatase (AlP) histochemical analysis. Calvarial osteoblasts were cultured with (con) or without 5α-dihydrotestosterone (DHT) (10-8 M) for 21 days. The cultures were evaluated by AlP histochemistry and von kossa silver staining on left. Quantification of bone nodules formed is shown on right. A Primary cultures from wild-type mice (n = 5). B Primary cultures from AR3.6-transgenic mice (n = 5). C Primary cultures from AR2.3-transgenic mice (n = 4 or 5). Representative images for combined AlP and von kossa staining are shown. Results are expressed as mean ± standard deviation (SD). A significant decrease in the number of bone nodules was observed with DHT addition. * p < 0.05; *** p < 0.001 compared to control (con) [138]. 8


FIGURE 15.8 Histological analysis of site-specific anabolic action in calvaria. Fine mapping of the calvarial response over the surface of the calvaria harvested from 2-month male WT and AR3.6-tg mice. Sagittal sections across frontal and parietal bones stained with hematoxylin and eosin stain (H-E) (A) and von gieson (B) indicate that in transgenic males only the frontal bone demonstrates an anabolic thickening (arrow), and not the parietal bones. Inset shows region of higher magnification image below. F indicates frontal bone, P indicates parietal bone. Total magnification 40 x, inset 100 x. Scale bar = 100 μm [110].

9


FIGURE 15.9 Androgen-mediated opposite effects of alkaline phosphatase (AlP) activity and mineralization during differentiation in cultures from AR3.6-tg fNCSC vs. pMSC. Cultures were induced toward the osteoblast lineage with osteoinductive media and osteoblastogenesis assessed by AlP staining, alizarin red S (AR-S) and von kossa staining. (A) Characterization of osteoblastogenesis using AlP, von kossa, and AR-S staining. Both fNCSC and pMSC cells were grown with osteoblastogenic medium at confluence. At day 10, cultures were stained and mineralized nodule formation was assessed by von Kossa over ALP staining. For mineral accumulation analysis, both fNCSC and pMSC cells were grown with osteoblastogenic medium for 14 days, and mineralization was assessed by alizarin red-S (AR-S) staining. Similar analysis at lower power in 12-well plates is shown in right panel. (B) AR-S quantitation after extraction. AR-S accumulation was expressed as fold vs. undifferentiated control. Data are expressed as mean ± standard deviation (SD) (n = 3). Two-way analysis of variance (ANoVA) for the effects of genotype and embryonic lineage showed a significant interaction, so t-test was used to determine significance. ** p < 0.01 vs. WT; ## p < 0.01 vs. fNCSC WT. osteoblast markers AlP (C) and osteocalcin (oC) (D) gene expression. levels were evaluated by real-time polymerase chain reaction (qPCR) using ribonucleic acid (RNA) isolated from mature mineralizing cells from both genotypes and both lineages grown in osteoblastogenic media for 10 days. Data are reported as mean ± standard deviation (SD) (n = 3). * p < 0.05; ** p < 0.01 compared to WT. uD: undifferentiated [110].

10


FIGURE 15.10 gene expression differences in transforming growth factor (TgF)-β/bone morphogenetic protein (BMP) signaling in cortical bone from androgen receptor (AR)-transgenic males. Multiple components of BMP signaling are influenced by androgen action, with expression of several BMP antagonists upregulated in bone. upregulated transcripts are shown in red; downregulated are green; straight lines indicate protein binding or interaction; arrows indicate activation; inhibition is shown as a T-shaped line [138].

11


FIGURE 15.11 Model for site-specific androgen action in skeletal tissues. The consequences of androgen action in stem cells, trabecular bone, and cortical sites vary depending on location and embryonic origin. AR: androgen receptor; DHT: 5α-dihydrotestosterone; MSC: mesenchymal stem cell.

12


FIGURE 15.12 Characterization of cortical bone formation in androgen receptor (AR)-transgenic (AR-tg) mice. Dynamic histomorphometric analysis was performed in cortical bone after fluorescent imaging microscopy in AR-tg males (n = 6–8). Mineralizing surface as a percent of bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR) at both the endocortical and periosteal surfaces were determined in wild-type (wt) and AR-tg mice. *p < 0.05 [19].

13

Chapter 15 Chapter 15 Androgens and Skeletal Biology: Basic Mechanisms Copyright © 2013 Elsevier...

Documents

Transcript of Chapter 15 Chapter 15 Androgens and Skeletal Biology: Basic Mechanisms Copyright © 2013 Elsevier...