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The enzyme 5α-reductase, which converts testosterone to dihydrotestosterone (DHT), performs key functions in the androgen receptor (AR) signaling pathway. The three isoenzymes of 5α-reductase identified to date are encoded by different genes: SRD5A1, SRD5A2, and SRD5A3. In this study, we investigated mechanisms underlying androgen regulation of 5α-reductase isoenzyme expression in human prostate cells. We found that androgen regulates the mRNA level of 5α-reductase isoenzymes in a cell type-specific manner, that such regulation occurs at the transcriptional level, and that AR is necessary for this regulation. In addition, our results suggest that AR is recruited to a negative androgen response element (nARE) on the promoter of SRD5A3 in vivo and directly binds to the nARE in vitro. The different expression levels of 5α-reductase isoenzymes may confer response or resistance to 5α-reductase inhibitors and thus may have importance in prostate cancer prevention.

Our previous work has provided strong evidence that the transcription factor SOX9 is completely needed for chondrogenic differentiation and cartilage formation acting as a "master switch" in this differentiation. Heterozygous mutations in SOX9 cause campomelic dysplasia, a severe skeletal dysmorphology syndrome in humans characterized by a generalized hypoplasia of endochondral bones. To obtain insights into the logic used by SOX9 to control a network of target genes in chondrocytes, we performed a ChIP-on-chip experiment using SOX9 antibodies.The ChIP DNA was hybridized to a microarray, which covered 80 genes, many of which are involved in chondrocyte differentiation. Hybridization peaks were detected in a series of cartilage extracellular matrix (ECM) genes including Col2a1, Col11a2, Aggrecan and Cdrap as well as in genes for specific transcription factors and signaling molecules. Our results also showed SOX9 interaction sites in genes that code for proteins that enhance the transcriptional activity of SOX9. Interestingly, a strong SOX9 signal was also observed in genes such as Col1a1 and Osx, whose expression is strongly down regulated in chondrocytes but is high in osteoblasts. In the Col2a1 gene, in addition to an interaction site on a previously identified enhancer in intron 1, another strong interaction site was seen in intron 6. This site is free of nucleosomes specifically in chondrocytes suggesting an important role of this site on Col2a1 transcription regulation by SOX9.Our results provide a broad understanding of the strategies used by a "master" transcription factor of differentiation in control of the genetic program of chondrocytes.

To study the structure/function relationship of the prosegment of aspartic proteinase, a putative proform of aspergillopepsin I (or proteinase B) from Aspergillus niger var. macrosporus was expressed by Escherichia coli, refolded in vitro, and purified. The conversion of the purified proenzyme (aspergillopepsinogen I, proproteinase B) into the active mature form occurred at pH ≤ 4.5 and was completely inhibited by pepstatin A, a specific inhibitor for aspartic proteinase, suggesting autoprocessing. The N-terminus of this mature form was Glu67 (numbering in preproform), which was different from the N-terminal Ser70 of native proteinase B although there was no significant difference in enzymatic activity. During the conversion, two intermediates were observed on SDS/PAGE, indicating a stepwise mechanism. The Lys56-Phe57 sequence seems to be a counterpart of the Lys-Tyr pair highly conserved in the prosequences of aspartic proteinases. When the mutant proenzyme (K56N), in which Lys56 was replaced with Asn by site-directed mutagenesis, was allowed to refold under various conditions, no significant potential activity could be obtained. Proproteinase B was also expressed by Bacillus brevis HPD31. This system required no in vitro refolding to obtain potentially active proenzyme, which was secreted into the culture medium (30-120 mg/l) and had the same properties with that obtained by the E. coli system. The K56N mutant prepared by this system also had a no potential activity, and was rapidly digested by incubation with native proteinase B, suggesting that the mutant did not fold correctly. On the other hand, the K56R mutant (Lys56→Arg) was potentially active. These results indicated that Lys56 is essential for the folding through electrostatic interaction with the catalytic Asp residues in the active site although it may be replaced with Arg. In the presence of a low concentration of pepstatin A, an incompletely processed form with N-terminal Ser53 was obtained. Further, the R52Q (Arg52→Gln) mutant showed no processing but was converted to the active mature form by incubation with the native enzyme. Therefore, the cleavage between Arg52 and Ser53 is considered to be the initial and essential step of the autoactivation. The R26Q, K27Q, R36Q, K40Q, R42Q, and K66Q mutants were also potentially active. K66Q mutant was processed to a form with N-terminal Ala55. [ABSTRACT FROM AUTHOR]

Induction of new bone formation is frequently seen in the bone lesions from prostate cancer. However, whether osteogenesis is necessary for prostate tumor growth in bone is unknown. Recently, 2 xenografts, MDA-PCa-118b and MDA-PCa-133, were generated from prostate cancer bone metastases. When implanted subcutaneously in severe combined immunodeficient (SCID) mice, MDA-PCa-118b induced strong ectopic bone formation whileMDA-PCa-133 did not. To identify the factors that are involved in bone formation, we compared the expression of secreted factors (secretome) from MDA-PCa-118b and MDA-PCa-133 by cytokine array. We found that the osteogenic MDA-PCa-118b xenograft expressed higher levels of bone morphogenetic protein BMP4 and several cytokines including interleukin-8, growth-related protein (GRO), and CCL2. We showed that BMP4 secreted from MDA-PCa-118b contributed to about a third of the osteogenic differentiation seen in MDA- PCa-118b tumors. The conditioned media from MDA-PCa-118b induced a higher level of osteoblast differentiation, which was significantly reduced by treatment with BMP4 neutralizing antibody or the small molecule BMP receptor 1 inhibitor LDN-193189. BMP4 did not elicit an autocrine effect on MDA-PCa-118b, which expressed low to undetectable levels of BMP receptors. Treatment of SCIDmice bearingMDA-PCa-118b tumors with LDN- 193189 significantly reduced tumor growth. Thus, these studies support a role of BMP4-mediated osteogenesis in the progression of prostate cancer in bone. Cancer Res; 71(15); 5194-203. ©2011 AACR. [ABSTRACT FROM AUTHOR]