Androgen Regulation of Murine P-Glucuronidase Expression: Identification and Characterization

One of the major features of P-glucuronidase (GUS) expression in inbred strains of the house mouse, Mus musculus, is the responsiveness of this enzyme to androgen stimulation in tubule cells of the kidney. Both GUS-specific and nonspecific mutations have been described which define genes that serve to control this response. During examination of the expression of GUS in the interbreeding subspecies, Mus hortulanus, a new GUS haplotype was uncovered that is characterized, in part, by a lack of GUS response to androgen stimulation in an apparently responsive kidney. Blot hybridization analyses of kidney RNA with a radiolabeled murine GUS cDNA shows this lack of response to be reflected in GUS mRNA levels. The difference in heat stability of GUS activity between M . hortulanus and a responsive inbred strain, ICWHa, was utilized to assess the contribution of each parent to kidney levels of GUS in androgen-treated and -untreated F1 progeny of these strains. The results, together with preliminary genetic studies, suggest that the element controlling this responsiveness (or the lack thereof) is cis-active and tightly linked to the GUS structural gene on chromosome 5. It is not known whether this element is identical to another GUS-specific, cis-active element, Gus-r, which also controls the androgen response of GUS in mouse kidney. T HE androgen responsiveness of kidney in laboratory mice and strains of the house mouse has been well characterized (OHNO et al. 1970; BARDIN et al. 1973; SCHWARZLOSE and HEIM 1973; SWANK, PAIGEN and GANSCHOW 1973). Androgenic steroids induce cellular hypertrophy and an increase in specific activities of several enzymes in the epithelial cells of proximal convoluted tubules. These responses are dependent upon the presence of functional androgen receptors (BARDIN et al. 1973). Analysis of the genetic control of enzyme expression in response to hormonal stimulation has been enhanced in recent years by the availability in inbred mice of natural variants of specific enzyme phenotypes (SWANK, PAIGEN and GANSCHOW 1973; PAIGEN, LABARCA and WATSON 1979; WATSON et al. 198 1 ; PALMER et al. 1983). Variation in both structural and regulatory determinants has provided a resource for initiating genetic and biochemical analyses of the mechanisms involved in the regulation of these enzymes. Murine P-glucuronidase (P-D-glucuronide glucuronohydrolase; EC 3.2.1.31) is one of the most extensively characterized genetic models for studying enzyme and gene regulation in mammals. Analysis of P-glucuronidase (GUS) expression in inbred strains of mice has uncovered natural variation in protein structure, intracellular localization, hormonal reguGenetics 119: 151-156 (May, 1988). lation and developmental accumulation (reviewed in PAIGEN 1979). Levels of GUS and its mRNA increase dramatically in mouse kidney following androgen administration. The magnitude of this response is controlled by a cis-active regulatory element, designated Gus-r (SWANK, PAIGEN and GANSCHOW 1973; PAIGEN, LABARCA and WATSON 1979; WATSON et al. 198 1 ; PALMER et al. 1983). Variants which define Gus-r differ in the initial lag period and in the extent of kidney GUS responsiveness to androgens (SWANK, PAIGEN and GANSCHOW 1973). This variation along with that of the GUS structural gene, Gus-S, partially define the two major GUS haplotypes, [Gus]” and [Gus]’. We are utilizing this and other genetic variation in GUS expression to identify DNA sequences which underlie androgen regulation. Additional GUS haplotypes have been identified in surveys of house mice trapped in diverse geographical locations. These mice typically interbreed with laboratory strains and share many properties of GUS expression. In general, the diverse samples of house mice frequently carry new alleles for GUS structure and regulation and thus represent new haplotypes (PAIGEN 1979; PFISTER et al. 1985). Nevertheless, all of the house mouse species examined uniformly show kidney responsiveness to androgen stimulation. Con152 S. D. Lund et al. versely, previous work on different Mus species which do not interbreed with laboratory strains suggests that the induction of kidney GUS in response to androgen is limited to the house mouse (SWANK et ul. 1978). We now extend these surveys of GUS expression to Mus hortulunus, a field mouse from central Europe which does not interbreed in the wild with the house mouse Mus musculus. However, F1 hybrids can be produced under laboratory conditions. We demonstrate that the androgen response of kidney GUS is absent in M . hortulunus, and that this lack of response is determined by a cis-acting genetic element closely associated with the GUS structural gene. MATERIALS AND METHODS Mice: C57BL/6J inbred mice and ICWHa random bred mice were obtained from the Jackson Laboratory (Bar Harbor, Maine). The wild-derived species, M . hortulanus, was isolated in southeastern Europe by R. SAGE and sent to Roswell Park Memorial Institute (Buffalo, New York). Outbred colonies of M . hortulanus from Pancevo, Yugoslavia, are currently maintained with a minimum of six to ten pairs of unrelated animals in each generation. Androgen treatment was achieved by subcutaneous implantation of a 30 mg testosterone pellet in the nape of the neck. Females 10 to 16 weeks of age were used in all experiments. M . hortulanus does not interbreed with the house mouse species M . musculus in the wild, but F, hybrids can be produced through artificial insemination. Female IClUHa mice were injected i.p. with 5 IU Pregnant Mare Serum (PMS from Sigma, St. Louis), 48 hr later with human gonadotropin (HCG from Sigma, St. Louis) and inseminated 12 hr later. Epididymides from adult (over 5 months old) male M . hortulanus were removed and placed into 1.5 ml Brinster's medium (from GIBCO, Grand Island, New York) in a watch glass. After removing excess fat, each epididymis was cut five to six times using superfine iris scissors to liberate the sperm. A 22-gauge deburred needle bent to a 75" angle attached to a 1-ml syringe was used to deliver 0.05 ml sperm into the vagina of an etherized mouse (as prepared above). The vagina was then plugged with a small wad of cotton. Litters, with an average size of 14, develop within a normal gestation period. Tissue preparation and GUS assay: Kidneys were homogenized with a Polytron (Brinkman Instruments) in 0.25 M sucrose and 0.02 M imidazole (pH 7.4) to make 10% (w/ v) homogenates. GUS activity was assayed using 4-methylumbelliferyl-P-D-glucuronic acid as a fluorometric substrate (LLSIS, TOMINO and PAIGEN 1976). All activities are expressed in kmol/hr/g wet weight of tissue at 37". Heat inactivation: Kidney homogenates were diluted 50-fold in 0.1 M sodium acetate (pH 5.0) for androgentreated Ha/ICR and C57BL/6J mice. Androgen-treated M . hortulanus and all untreated kidney homogenates were diluted oneto twofold in 0.1 M sodium acetate (pH 5.0). Aliquots of 250 ~1 of undiluted and/or diluted kidney homogenates were incubated at 71.5" for various times. The inactivation reaction was stopped by immersing the tubes in ice water, Remaining GUS activity was determined using the fluorometric substrate assay on 50 ~1 of the heattreated samples. RNA isolation and hybridization analysis: Total cellular RNA was isolated by the guanidine-thiocyanate procedure TABLE 1 Lack of androgen response of kidney GUS activity levels in M. hortulanus Glucuronidase activity levels (pmolihrlg) Days of treatment M . hortulanw hortulanw) ICR C57BU6J FI (ICR X M . 0 7 1 . 8 2 0 . 1 2 . 5 5 0 . 0 4 . 0 t 0 . 1 3 . 2 e 0 . 3 2.8 2 0.1 23.9 f 0.7 40.7 ? 0.1 39.9 5 2.0 14 2.5 5 0.1 36.3 5 0.4 41.4 5 0.8 39.2 t 4.0 ICR and C57BIJ6J mice are [ C W ] ~ haplotype. A minimum of four individual female kidneys were used in each group. The values are represented as mean 2 the standard error. of CHIRGWIN and co-workers (1979) modified for use with tissues. For blot hybridization analysis, total kidney RNA was electrophoresed in a 1% agarose gel containing 18% formaldehyde and transferred to Zetabind (AMF Cuno). The hybridization probes were labelled with 32P using the random primer method of FEINBERG and VOGELSTEIN (1983). The probe used in the hybridization analysis was a 1.5 kb PstI-Hind111 fragment of the cDNA clone, pGUS-1 (PALMER et al. 1983). RNA dot blot analysis was performed using a filtration manifold (Schleicher and Schuell, Inc.). RNA samples (10 Fg) were denatured in a solution of 3 parts 2 0 ~ SSC (3.7 M NaCl, 0.375 M sodium citrate) and 2 parts 3 7 4 formaldehyde. Serial dilutions were applied to Zetabind and probed with pGUS-1 under the same conditions used for blot hybridization.