Androgen-regulated expression of a novel member of the aldo-keto reductase superfamily in regrowing rat prostate.

The rat prostate is dependent on androgen for normal growth and differentiation. In addition, the organ undergoes rapid cell death upon withdrawal of androgen on castration, and the atrophied tissue is capable of regrowth after androgen replacement in adult animals. In our search for novel factor(s) that participate in this androgen-induced proliferation of adult rat prostate cells, we have generated a complementary DNA (cDNA) library enriched in cDNAs transiently up-regulated after androgen stimulation in castrated rat ventral prostate using a PCR-based subtractive hybridization technique. Sequence analysis of about one hundred clones in the library showed that approximately 70% of them are identical or closely related to genes of known function, the remaining ones showing no or very low similarity to any genes characterized previously. Among the former a new member of the rat aldo-keto reductase superfamily that is closely related to aflatoxin, B1 aldehyde reductase has been identified. The newly identified protein (androgen-inducible aldehyde reductase, AIAR) and rat aflatoxin B1 aldehyde reductase (AFAR) exhibit 80% amino acid sequence homology. The enzymatic activity toward 4-nitrobenzaldehyde of recombinant AIAR expressed in Escherichia coli was about 16% of that of rat AFAR. Northern blot analysis revealed AIAR expression in various adult rat tissues in addition to the ventral and dorsolateral prostates, which differs from the highly restricted expression of AFAR in the kidney and liver. The AIAR messenger RNA (mRNA) content of the ventral prostate was low in normal and castrated rats, transiently increased after androgen administration to castrated rats, attaining a peak 12-24 h after the treatment. Although the physiological substrate(s) of AIAR has not been identified, the current results suggest that AIAR expression is associated with some growth-related processes in regrowing rat prostate.

Role of nitric oxide in regulation of renal sympathetic nerve activity during hemorrhage in conscious rats.

The effect of inhibition of nitric oxide (NO) synthesis on the responses of blood pressure (BP), heart rate (HR), and renal sympathetic nerve activity (RSNA) during hemorrhaging was examined with the use of an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), in conscious rats. In the 0.9% saline group, hemorrhage (10 ml/kg body wt) did not alter BP but significantly increased HR and RSNA by 88 +/- 12 beats/min and 67 +/- 12%, respectively. Intravenous infusion of L-NAME (50 microg. kg(-1). min(-1)) significantly attenuated these tachycardic and sympathoexcitatory responses to hemorrhage (14 +/- 7 beats/min and 26 +/- 12%, respectively). Pretreatment of L-arginine (87 mg/kg) recovered the attenuation of HR and RSNA responses induced by L-NAME (92 +/- 6 beats/min and 64 +/- 10%, respectively). L-NAME by itself did not alter the baroreceptor reflex control of HR and RSNA. Hemorrhage increased the plasma vasopressin concentration, and its increment in the L-NAME-treated group was significantly higher than that in the 0.9% saline group. Pretreatment with the vascular arginine vasopressin V(1)-receptor antagonist OPC-21268 (5 mg/kg) recovered the attenuation of RSNA response induced by L-NAME (54 +/- 7%). These results indicate that NO modulated HR and RSNA responses to hemorrhage but did not directly affect the baroreceptor reflex arch. It can be assumed that NO modulated the baroreflex function by altering the secretion of vasopressin induced by hemorrhage.

Collagen-binding growth factors: production and characterization of functional fusion proteins having a collagen-binding domain.

The autocrine/paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF/CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB/c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.

Isolation and characterization of invasive and noninvasive variants of a rat bladder tumor cell line.

We isolated, in vitro, spontaneous variants of the rat bladder tumor NBT-II cell line with a distinctive morphology. Of five sublines obtained, three (NBT-L1, L2a and L2b) exhibited an elongated shape and moderate to high invasive activity in vitro. The other two sublines (NBT-T1 and T2) formed tight colonies and exhibited very low or negligible invasive activity. The contents of mRNAs coding for E-cadherin and cadherin-associated molecules (alpha-catenin and beta-catenin) were not correlated with the invasive activity of the cells. However, the expression level of the E-cadherin protein, but not those of catenins, was lower in invasive cells (NBT-L1, L2a and L2b) than in noninvasive cells (NBT-T1 and T2). Analysis of mRNAs coding for several growth factors and their receptors showed that the transforming growth factor alpha mRNA content in invasive cells was higher than that in noninvasive cells, and that the content of epidermal growth factor receptor mRNA was low in NBT-T2. Although NBT-II is known to acquire a fibroblastic appearance and cell motility in response to several growth factors, the conditioned media of the invasive sublines hardly affected the morphology or motility of noninvasive cells. These results indicate that the decreased E-cadherin expression is closely associated with the transition from the noninvasive to the invasive phenotype of the bladder tumor cells, and that a post-transcriptional process is important in the control of E-cadherin expression in the cells. These sublines may be useful as models for studies on the progression of bladder tumors.

A novel splicing junction mutation in the gene for the steroidogenic acute regulatory protein causes congenital lipoid adrenal hyperplasia.

Congenital lipoid adrenal hyperplasia (lipoid CAH) is a relatively common genetic disorder of adrenal and gonadal steroidogenesis and is the most severe form of CAH. As typical affected individuals cannot produce any steroid hormones or can only produce low levels of steroid hormones in the adrenals and gonads, including glucocorticoids, mineralcorticoids, and sex steroids, a genetic defect in the cholesterol side-chain cleavage enzyme, cytochrome P450scc (CYPXIA1), has been postulated to be the cause of their insufficient production to date. Recently, Lin and co-workers proved a link between mutations of the steroidogenic acute regulatory protein (StAR) gene and the lipoid CAH phenotype. Therefore, we investigated both the cytochrome P450scc and StAR genes in a Korean family with a fairly mild form of lipoid CAH to identify the mutation(s) causing this disease. The result was that no mutations could be found in the two genes, except for a thymine (T) insertion into intron 2 of the StAR gene, 3 bp from the splice donor site of exon 2. PCR-amplified StAR genes from a normal subject and the patient were cloned into an expression vector and then introduced into COS-7 cells. Northern blot and reverse transcriptase-PCR analyses indicated that the StAR messenger ribonucleic acid derived from the vector with the normal StAR gene spliced exons 2 and 3 correctly, whereas most, but not all, StAR messenger ribonucleic acid derived from the vector with the T-inserted StAR gene could not remove intron 2. We concluded from these results that the T insertion into the StAR gene accounts for the lipoid CAH phenotype in this patient.

Changes in gene expression of growth factors and their receptors during castration-induced involution and androgen-induced regrowth of rat prostates.

To find candidates for the mediator of the growth-promoting action of androgen in rat prostates, the changes in the steady-state levels of mRNAs coding for several growth factors and their receptors were examined by Northern blot analysis during castration-induced involution, and subsequent regrowth induced by androgen in the ventral and dorsolateral lobes. The changes in the growth factor systems and a typical secretory protein in the ventral lobe were similar to, but more prominent than, those in the dorsolateral lobe, showing the higher androgen dependency of the ventral lobe. Among the growth factors and their receptors investigated, only epidermal growth factor (EGF) showed apparent positive androgen dependency: EGF mRNA content in the ventral lobe decreased to about 30% of the normal level within 24 hr after castration, and increased, attaining about 200-300% of the normal level 3-5 days after androgen administration to castrated rats. mRNAs coding for all other factors examined, i.e., transforming growth factor-alpha (TGF-alpha), EGF receptor, basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1, TGF-beta1, TGF-beta type II receptor, hepatocyte growth factor (HGF), and c-MET/HGF receptor, increased after castration in greater or lesser degree, and after a brief pause or a decrease some of them increased again attaining a second peak 3-5 days after androgen replacement. The second increase was evident in TGF-alpha, EGF receptor, KGF, and c-MET mRNAs. These results indicate the possibility that multiple growth factor-receptor systems participate in the androgen-dependent regrowth of castrated rat prostates.

Enhanced gene expression of transforming growth factor-alpha and c-met in rat urinary bladder cancer.

To investigate the roles of growth factors in bladder cancer, changes in the expression of messenger RNAs (mRNAs) for several growth factors and their receptors were examined during rat bladder carcinogenesis induced with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Northern blot analysis showed that the contents of mRNAs for transforming growth factor-alpha (TGF-alpha) and c-met/hepatocyte growth factor (HGF) receptor increased with BBN treatment. Epidermal growth factor (EGF) receptor mRNA was hardly affected by the treatment; while mRNA for fibroblast growth factor (FGF) receptor 1 and transforming growth factor-beta (TGF-beta) type II receptor decreased with BBN treatment. A rat bladder tumor cell line, NBT-II, expressed both TGF-alpha and c-met mRNAs, and HGF showed apparent scattering and growth-stimulating effects on the cells. These results indicate the possibility that TGF-alpha produced by a bladder cancer, in addition to urinary EGF, plays a role in the development of bladder cancer, and that enhanced cell motility due to activation of the c-met/HGF receptor participates in the invasion and metastasis of the cancer cells.

Identification of probasin-related antigen as cystathionine gamma-lyase by molecular cloning.

We reported previously that a monoclonal antibody against probasin (rat prostatic secretory protein) recognizes a 40-kDa protein localized in rat liver and kidney. The protein (probasin-related antigen, PRB-RA) may participate in a specific differentiated function of these tissues. To clarify the molecular nature of PRB-RA, a series of cDNA clones coding for the protein were isolated from a rat liver expression library using an affinity-purified polyclonal antibody. The amino acid sequence deduced from the determined cDNA sequence included sequences identical with those of proteolytic fragments of PRB-RA, which covered about 70% of the deduced sequence. Northern blot hybridization of poly(A)+ RNA isolated from rat tissues showed the presence of predominant and minor mRNA species of about 2.0 and 4.3 kilobases, respectively, in the liver and kidney. A sequence homology search revealed that PRB-RA is almost completely identical to rat cystathionine gamma-lyase (cystathionase) and that it does not show overall homology with probasin. Three candidates for an epitope common to probasin and PRB-RA were found on close examination of the amino acid sequences of the two proteins. A synthetic peptide, TYFRRI, corresponding to one of the candidates, neutralized the reactivity of the anti-probasin monoclonal antibody to both probasin and PRB-RA on Western blot analysis. These results show that PRB-RA/cystathionase is neither structurally nor functionally related to probasin except for a common epitope and that cystathionase, a cystein-producing enzyme, is localized in urinary tubular epithelial cells in a highly restricted region of the kidney in addition to in liver parenchymal cells.

An anti-probasin monoclonal antibody recognizes a novel 40-kDa protein localized in rat liver and a specific region of kidney urinary tubule.

Immunoblotting with a monoclonal antibody against probasin (rat prostatic secretory protein) showed that a 40-kDa protein antigenically related to probasin was localized in rat liver and kidney. The contents of probasin in these organs were negligible. Immunostaining revealed that the 40-kDa protein (probasin-related antigen: PRB-RA) was expressed in the liver parenchymal cells and the kidney urinary tubular epithelial cells in outer stripe. The content of PRB-RA in the kidney was low during 0 to 2 weeks of age, then rapidly increased about 10-fold from 2 to 8 weeks of age. The content in the liver increased about 2-fold during the period, reaching a value of 10-12 ng/micrograms protein, which was ten times higher than that in the kidney. PRB-RA was purified from rat liver by ion-exchange chromatography, gel filtration and fast protein liquid chromatography on a hydroxyapatite column. The purified protein formed insoluble aggregates in the absence of a detergent, and it had a blocked amino terminal. The amino acid sequence of a peptide generated by tryptic digestion of alkylated PRB-RA was determined. Computer analysis showed that there was no protein having a significant homology with the peptide. These results indicate that a novel 40-kDa protein with a structural similarity to probasin is localized in rat liver and kidney, and might bear a function specific to these organs.