Daniel A. Meier

Characteristics of FGF-receptors Expressed by Stromal and Epithelial Cells Cultured from Normal and Hyperplastic Prostates

Three fibroblast growth factors (FGFs), acidic FGF (FGF1), basic FGF (FGF2), and keratinocyte growth factor (FGF7) have been identified in prostate. To understand how FGFs regulate growth of the prostate, and to determine if regulation is altered in benign prostatic hyperplasia (BPH), the mitogenic potential of FGFs, receptor binding, and FGF-receptor (FGFR) gene expression of stromal (PS) and epithelial cells (PE) cultured from normal human prostate and BPH where determined. FGF1 and FGF2, but not FGF7, were mitogens for PS. FGF1 and FGF7 were potent mitogens for PE, but FGF2 was a weak mitogen for these cells. Both PS and PE exhibited high affinity binding (pM K) of iodinated-FGF2. The K was 4-fold and 12-fold higher for PS than for PE cultured from normal prostate and BPH, respectively. Northern analysis indicated that PS, but not PE, expressed FGFR type 1 (FGFR1) mRNA. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate FGFR type 2 (FGFR2) expression. The size of amplified DNA fragments, and nucleotide sequences, indicated that PS also expressed transcripts for the exon IIIc RNA splice variant of FGFR2. A RT-PCR product with the FGFR2 exon IIIb nucleotide sequence joined with the exon IIIc sequence was amplified with poly A+ RNA from PE and primers spanning both exons. Thus, PE did not alternatively splice mRNA for FGFR2 exon IIIb and exon IIIc. No differences in the mitogenic potential of FGFs, receptor binding (K or number of sites), or FGFR gene expression were found in cells cultured from normal prostate and BPH.

Regulation of basic fibroblast growth factor expression by transforming growth factor beta in cultured human prostate stromal cells

Basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGFβ1) are potential autocrine growth regulators of the prostatic stroma, and therefore may play a role in the development of benign prostatic hyperplasia (BPH). We reported [Story et al.: Prostate 22:183–197, 1993] that TGFβ1 increased bFGF and bFGF mRNA expression in cultured human prostate stromal cells (PS). The current study extends those studies and investigates the mechanism by which TGFβ1 upregulates the level of bFGF mRNA. A solution hybridization assay was used to quantitate bFGF mRNA. Treatment of PS for 6 hr with TGFβ1 (1 ng/ml) maximally stimulated bFGF mRNA expression. TGFβ2 and TGFβ3 were similarly active in upregulating bFGF mRNA. TGFβ1 or cycloheximide each increased bFGF mRNA about 3‐fold. The effect of these agents was not additive. This suggested that a labile protein was involved in processing bFGF mRNA. Determination of message stability indicated that the half‐life of bFGF mRNA in TGFβ1‐treated PS was 6.8 hr, as compared to 4.3 hr in untreated cells. The data indicated that posttranscriptional mechanisms, that increased message stability were, at least in part, responsible for upregulation of bFGF mRNA by TGFβ1 in PS. Our studies suggest that growth of the prostatic stroma is regulated by the interaction of members of two families of growth modulators, bFGF and TGFβ. It remains to be determined if an imbalance in this system in favor of stroma hyperplasia plays a role in the development of BPH.

Divergence between GLUT4 mRNA and protein abundance in skeletal muscle of insulin resistant rats

Hyperglycemia and skeletal muscle insulin resistance coexist in uncontrolled type 2 diabetes mellitus. Similar defects in insulin action were observed in glucose-infused, normal rats, a model of glucose toxicity. In these rats insulin-stimulated glucose uptake by skeletal muscle was decreased due to a post-receptor defect. We investigated whether the impaired glucose uptake resulted from a decrease in the abundance of the predominant muscle glucose transporter (GLUT4) mRNA and/or protein. GLUT4 protein abundance in the hyperglycemic rats was not different from the control group despite a 50% decrease in muscle glucose uptake. GLUT4 mRNA abundance was 2.5-fold greater in the hyperglycemic rats as compared to the control animals. We conclude that the coexistence of hyperglycemia and hyperinsulinemia results in (1) a defect in GLUT4 compartmentalization and/or functional activity and (2) a divergence between GLUT4 mRNA levels and translation.

Quantitation of GLUT1 and GLUT4 mRNA using a solution hybridization assay

The development of a solution hybridization assay for detecting GLUT1 and GLUT4 mRNA is described. The details of this assay are described in which copy RNA is used to quantitate messenger RNA in total RNA samples. This solution hybridization assay is highly specific and reproducible and is significantly more sensitive than Northern blotting. Since GLUT mRNAs can be quantitated in as little as 25 mg tissue, this technique is essential when the supply of tissue is limited. Furthermore, the elimination of gel-based separation techniques allows for mRNA quantitation in several hundred samples within two days following isolation of samples.

Vasoactive Intestinal Polypeptide Antiserum Affects Rat Prolactin mRNA in 40-Day but Not 110-Day Diethylstilbestrol-Induced Prolactinoma Tissue

Experiments were designed to examine whether vasoactive intestinal polypeptide (VIP), a known stimulator of basal prolactin (PRL) secretion, regulates PRL gene expression in the rat diethylstilbestrol (DES)-induced prolactinoma model. The VIP-induced increase in PRL release could result from increased PRL synthesis and/or decreased PRL degradation. Male Fischer 344 rats were implanted with 10 mg DES pellets 40 or 110 days prior to obtaining the anterior pituitary glands for cell dispersal. Cells were incubated in 1:10 normal rabbit serum or VIP antiserum (AVIP). After incubation, cells were pelleted, washed, and pooled for total nucleic acid extraction. The rat PRL (rPRL) mRNA abundance was quantitated using a solution hybridization/ribonuclease protection assay. Supernatant was collected and analyzed for PRL content using radioimmunoassay. Results from this experiment reveal partial immunoneutralization of intrapituitary VIP significantly decreased PRL secretory rate by rapid reduction in rRPL mRNA in the 40-day tumors. However, in the 110-day tumors the rPRL mRNA steady-state levels were unchanged but the basal release of PRL continued to be decreased by AVIP. These results indicate VIP exerts its effects on PRL secretion through at least two mechanisms.