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The binding of human sex hormone-binding globulin (SHBG) to a human prostatic cancer cell line (LNCaP) and the results of that binding were examined. Membranes derived from LNCaP cells bound unliganded SHBG on two sets of sites whose affinities were: Ka1 = 3.1 +/- 1.6 x 10(10) M-1 and Ka2 = 8.7 +/- 4.3 x 10(6) M-1. Intact cells also bound SHBG, but even after 6 h, less than 10% of specifically bound SHBG was internalized. This observation speaks against a role for the membrane binding of SHBG in steroid transport across cell membranes. When LNCaP cells were prebound with SHBG, addition of dihydrotestosterone or estradiol resulted in a dose-dependent increase in intracellular cAMP. SHBG in the absence of steroids or dihydrotestosterone in the absence of SHBG was without effect. 2-Methoxyestradiol, a steroid metabolite without biological activity, but which binds to SHBG more tightly than does estradiol, was also without effect. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.

Sex hormone-binding globulin (SHBG) is a plasma protein synthesized and secreted by the liver. Its initial description stemmed from its ability to bind estrogens and androgens and its capacity to regulate the free concentration of the steroids that bind to it. Additionally, it participates in signal transduction for certain steroid hormones at the cell membrane. It binds with high affinity to a specific membrane receptor (R SHBG ) in prostate stromal and epithelial cells, wherein the SHBG⅐R SHBG complex forms. An appropriate steroid binds to this complex and results in increases of intracellular cAMP. These two disparate functions of SHBG, regulation of the concentration of free steroids in plasma and signal transduction in selected tissues, raise the question of how its synthesis and secretion might be regulated so as to best perform these two disparate functions. In this paper we demonstrate that SHBG is produced in human prostate cancer cell lines (LNCaP, DU 145, and PC 3) as well as in cultured human prostate epithelial and stromal cells. In addition, in tissue sections of human prostate, we demonstrate the presence of SHBG (immunocytochemistry) and SHBG mRNA (in situ hybridization). These observations are consistent with the hypothesis that SHBG, destined to participate in signaling at the cell membrane, is locally regulated and produced.The hepatically derived (1) plasma protein, sex hormonebinding globulin (SHBG) 1 has two apparently disparate functions. It is the major regulator of free estrogens and androgens in plasma (2-4); it is an initiating component of a signaling system for estrogens and androgens at the cell membrane that functions via a G protein (5) and cAMP (6 -9). The details of the working of these systems generate a model that is in need of clarification in a number of crucial respects. In this paper we present data that deal with the source of the SHBG for signaling at the cell membrane. These data support a model in which the source of SHBG for signaling is not the plasma, but rather the target cells themselves.

Androgens are widely acknowledged to be central to the pathogensis of benign prostatic hypertrophy (BPH). However (GIBCO) with 5% fetal bovine serum containing penicillin (100 units/ml), streptomycin sulfate (100 ,ug/ml), and amphotericin (0.25 gg/ml) and was incubated for 2-3 days. It was then minced into 1-mm3 portions and transferred to 16-mm wells in serum-free medium (0.5 ml of RPMI 1640) for about 18 hr before the start of an experiment. All additions (SHBG, steroids, controls) were made in serum free RPMI 1640.Cell culture. Fibroblast-enriched cultures (17) were prepared from surgical specimens obtained as above. Fragments (1-3 mm3) were placed in 25 ml of digestion buffer (10 mM Hepes/142 mM NaCl/6.7 mM KC1, pH 7.4) and stirred for 30 min at 370C in an atmosphere of 95% air/5% CO2. After discarding the supernatant, fresh digestion buffer supplemented with 1% collagenase, 0.67 mM CaCl2, 20 mM dextrose, and 0.05% DNase was added to the tissue for 30 min at 370C. The supernatant was discarded again and the digestion was repeated twice. The cells released after the second and third digestion were collected, washed twice, and plated in fresh attachment medium (Ham's F12 supplemented with 5% fetal bovine serum). The medium was changed after 7 days and every 3-4 days thereafter. Three to four weeks after the original isolation, >95% of the cells had the typical morphology of fibroblasts. About 18 hr before the beginning of an experiment, cells were placed in serum-free medium (Ham's F12), and detached with a nonenzymatic cell dissociation solution (Sigma). They then were washed once and suspended (0.2-0.5 mg of protein per ml) in serum-free medium containing 50 nM SHBG. Prostatic epithelial cells were provided by D. M. Peehl (Stanford University) and were isolated as described by her (18). They underwent the same experimental protocol as the fibroblasts.Abbreviations: BPH, benign prostatic hypertrophy; SHBG, sex hormone-binding globulin. 5402The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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