Role of the cytokine-inducible SH2 protein CIS in desensitization of STAT5b signaling by continuous growth hormone.

Growth hormone (GH)-inducible suppressors of cytokine signaling (SOCS/CIS proteins) inhibit GH receptor (GHR) signaling to STAT5b via phosphotyrosine-dependent binding interactions with the tyrosine kinase JAK2 (SOCS-1) and/or the cytoplasmic tail of GHR (CIS and SOCS-3). Presently, we investigate the mechanism of CIS inhibition and CIS's role in down-regulating GHR-JAK2 signaling to STAT5b in cells exposed to GH continuously. CIS is shown to inhibit GHR-JAK2 signaling by two distinct mechanisms: by a partial inhibition that is decreased at elevated STAT5b levels and may involve competition between CIS and STAT5b for common GHR cytoplasmic tail phosphotyrosine-binding sites; and by a time-dependent inhibition, not seen with SOCS-1 or SOCS-3, that involves proteasome action. Investigation of the latter mechanism revealed that GH stimulates degradation of CIS, but not SOCS-3. The proteasome inhibitor MG132 blocked this protein degradation and also blocked the inhibitory action of CIS, but not that of SOCS-1 or SOCS-3, on STAT5b signaling. Proteasome-dependent degradation of CIS, most likely in the form of a (GHR-JAK2)-CIS complex, is therefore proposed to be an important step in the time-dependent CIS inhibition mechanism. Finally, the down-regulation of GHR-JAK2 signaling to STAT5b seen in continuous GH-treated cells could be prevented by treatment of cells with the proteasome inhibitor MG132 or by expression of CIS-R107K, a selective, dominant-negative inhibitor of CIS activity. These findings lead us to propose that the cytokine signaling inhibitor CIS is a key mediator of the STAT5b desensitization response seen in cells and tissues exposed to GH chronically, such as adult female rat liver.

SOCS/CIS protein inhibition of growth hormone-stimulated STAT5 signaling by multiple mechanisms.

The inhibition of growth hormone (GH) signaling by five members of the GH-inducible suppressor of cytokine signaling (SOCS/CIS) family was investigated in transfected COS cells. Complete inhibition of GH activation of the signal transducer STAT5b and STAT5b-dependent transcriptional activity was observed upon expression of SOCS-1 or SOCS-3, while partial inhibition (CIS, SOCS-2) or no inhibition (SOCS-6) was seen with other SOCS/CIS family members. SOCS-1, SOCS-2, SOCS-3, and CIS each strongly inhibited the GH receptor (GHR)-dependent tyrosine phosphorylation of JAK2 seen at low levels of transfected JAK2; however, only SOCS-1 strongly inhibited the GHR-independent tyrosine phosphorylation of JAK2 seen at higher JAK2 levels. To probe for interactions with GHR, in vitro binding assays were carried out using glutathione S-transferase-GHR fusion proteins containing variable lengths of GHR's COOH-terminal cytoplasmic domain. CIS and SOCS-2 bound to fusions containing as few as 80 COOH-terminal GHR residues, provided the fusion protein was tyrosine-phosphorylated. By contrast, SOCS-3 binding required tyrosine-phosphorylated GHR membrane-proximal sequences, SOCS-1 binding was tyrosine phosphorylation-independent, and SOCS-6 did not bind the GHR fusion proteins at all. Mutation of GHR's membrane-proximal tyrosine residues 333 and 338 to phenylalanine suppressed the inhibition by SOCS-3, but not by CIS, of GH signaling to STAT5b. SOCS/CIS proteins can thus inhibit GH signaling to STAT5b by three distinct mechanisms, distinguished by their molecular targets within the GHR-JAK2 signaling complex, as exemplified by SOCS-1 (direct JAK2 kinase inhibition), SOCS-3 (inhibition of JAK2 signaling via membrane-proximal GHR tyrosines 333 and 338), and CIS and SOCS-2 (inhibition via membrane-distal tyrosine(s)).

Thyroid regulation of NADPH:cytochrome P450 oxidoreductase: identification of a thyroid-responsive element in the 5'-flank of the oxidoreductase gene.

The current study demonstrates that T3-activated transcription of the NADPH:cytochrome P450 oxidoreductase (P450R) gene is dependent on the thyroid hormonal status of the animal, with both transcriptional and post-transcriptional pathways being important in regulating the cellular P450R mRNA level. The region required for transcriptional activation of the P450R gene by T3 has been identified. Nuclear run-on experiments demonstrated that the effects of T3 on P450R transcription are dependent on thyroid status, with a transcriptional enhancement obtained in T3-treated hypothyroid rat liver (1.8-fold increase) but not in T3-treated euthyroid animals. Transient cotransfection of P450R promoter/chloramphenicol acetyl transferase (CAT) constructs and the thyroid hormone receptor beta1 (TR beta1) expression plasmid into rat hepatoma H4IIE cells resulted in a 2.4-fold induction of promoter activity that was both T3 and TR beta1 dependent. Analysis of promoter deletion constructs identified a P450R-thyroid response region (P450R-TRE; bases, -564 to -536) containing three imperfect direct repeats of the thyroid response motif, AGGTCA. Mutational analysis further established that T3 induction was dependent only on the upstream direct repeat, having the sequence AGGTGAgctgAGGCCA. Footprint analysis showed that all three motifs were protected by proteins present in rat liver nuclear extracts, and a direct interaction between P450R-TRE and T3 receptors TR alpha1 and TR beta1 was demonstrated by gel-shift analysis. In vitro binding studies with P450R-TRE revealed the formation of heterodimeric complexes when TR alpha1 was coincubated with either the retinoic X receptor alpha or nuclear extract from rat liver, COS, or H4IIE cells. In addition, placement of the P450R-TRE upstream of the T3-nonresponsive heterologous thymidine kinase promoter resulted in a 2.7-fold transcriptional enhancement that was both T3 and TR beta1 dependent. Previous studies have demonstrated that T3 augments P450R mRNA levels approximately 20-30-fold and approximately 12-fold, respectively, in hypothyroid and euthyroid rats. Hence, for the hypothyroid state, transcriptional and post-transcriptional events contribute to the T3-induced mRNA increases; however, the marked increase in message level in T3-treated euthyroid animals depends primarily on post-transcriptional pathways.

Interaction of growth hormone-activated STATs with SH2-containing phosphotyrosine phosphatase SHP-1 and nuclear JAK2 tyrosine kinase.

Growth hormone (GH) rapidly stimulates tyrosine phosphorylation followed by serine/threonine phosphorylation of multiple cytoplasmic STAT transcription factors, including one, STAT5b, that is uniquely responsive to the temporal pattern of plasma GH stimulation in rat liver and is proposed to play a central role in the activation of male-expressed liver genes by GH pulses in vivo (Waxman, D. J., Ram, P. A., Park, S. H., and Choi, H. K. (1995) J. Biol. Chem. 270, 13262-13270). We now show that JAK2, the GH receptor-associated tyrosine kinase, is present both in the cytosol and in the nucleus in cultured liver cells and in rat liver in vivo and that GH-activated STAT3 but not STAT5b becomes associated with nuclear JAK2. GH is also shown to activate by 3-4-fold SHP-1, a phosphotyrosine phosphatase that contains two src homology 2 (SH2) domains. GH also induces nuclear translocation and binding of SHP-1 to tyrosine-phosphorylated STAT5b, suggesting that this GH-activated phosphatase may play a role in dephosphorylation leading to deactivation of nuclear STAT5b following the termination of a plasma GH pulse in male rat liver in vivo. No such association of SHP-1 with GH-activated STAT3 was detected, a finding that could help explain the marked desensitization of STAT3, but not STAT5b, to subsequent GH pulses following an initial GH activation event.

Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression.

The signal transducer and activator of transcription, STAT5b, has been implicated in signal transduction pathways for a number of cytokines and growth factors, including growth hormone (GH). Pulsatile but not continuous GH exposure activates liver STAT5b by tyrosine phosphorylation, leading to dimerization, nuclear translocation, and transcriptional activation of the STAT, which is proposed to play a key role in regulating the sexual dimorphism of liver gene expression induced by pulsatile plasma GH. We have evaluated the importance of STAT5b for the physiological effects of GH pulses using a mouse gene knockout model. STAT5b gene disruption led to a major loss of multiple, sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression were decreased to wild-type female levels in STAT5b-/- males, while female-predominant liver gene products were increased to a level intermediate between wild-type male and female levels. Although these responses are similar to those observed in GH-deficient Little mice, STAT5b-/- mice are not GH-deficient, suggesting that they may be GH pulse-resistant. Indeed, the dwarfism, elevated plasma GH, low plasma insulin-like growth factor I, and development of obesity seen in STAT5b-/- mice are all characteristics of Laron-type dwarfism, a human GH-resistance disease generally associated with a defective GH receptor. The requirement of STAT5b to maintain sexual dimorphism of body growth rates and liver gene expression suggests that STAT5b may be the major, if not the sole, STAT protein that mediates the sexually dimorphic effects of GH pulses in liver and perhaps other target tissues. STAT5b thus has unique physiological functions for which, surprisingly, the highly homologous STAT5a is unable to substitute.

Peroxisome proliferator-activated receptor alpha required for gene induction by dehydroepiandrosterone-3 beta-sulfate.

Peroxisome proliferator-activated receptor alpha (PPAR alpha) mediates the effects of foreign chemical peroxisome proliferators on liver and kidney, including the induction of peroxisomal, mitochondrial, and microsomal enzymes involved in beta-oxidation of fatty acids. However, the role of this receptor in the peroxisome proliferative effects of the endogenous steroid dehydroepiandrosterone (DHEA) is not known. DHEA-3 beta-sulfate fd(DHEA-S) is shown to induce a liver peroxisome proliferative response in rats in vivo at a dose at which DHEA is much less active, which is consistent with cultured hepatocyte studies indicating a requirement for sulfation for the activation of DHEA. Transient transfection experiments demonstrated that in contrast to the prototypic foreign chemical peroxisome proliferator pirinixic acid, DHEA-S and its 17 beta-reduced metabolite, 5-androstene-3 beta, 17 beta-diol-3 beta-sulfate, are inactive in mediating trans-activation by PPAR alpha in COS-1 cells. Two other mammalian PPAR isoforms, gamma and delta/Nucl, were also inactive with respect to DHEA-S trans-activation. To test whether PPAR alpha mediates peroxisomal gene induction by DHEA-S in intact animals, we administered DHEA-S or clofibrate to mice lacking a functional PPAR alpha gene. Both peroxisome proliferators markedly increased hepatic expression of two microsomal cytochrome P450 4A proteins as well as six mRNAs known to be associated with the peroxisomal proliferative response in wild-type mice. In contrast, neither DHEA-S nor clofibrate induced these hepatic proteins and mRNAs in PPAR alpha-deficient mice. Clofibrate-induced expression of kidney CYP4A mRNAs was also blocked in the PPAR alpha gene knockout mice. Thus, despite its unresponsiveness to steroidal peroxisome proliferators in transfection assays, PPAR alpha is obligatory for DHEA-S-stimulated hepatic peroxisomal gene induction. DHEA-S, or one of its metabolites, may thus serve as an important endogenous regulator of liver peroxisomal enzyme expression via a PPAR alpha-mediated pathway.

Growth hormone activation of Stat 1, Stat 3, and Stat 5 in rat liver. Differential kinetics of hormone desensitization and growth hormone stimulation of both tyrosine phosphorylation and serine/threonine phosphorylation.

Intermittent plasma growth hormone (GH) pulses, which occur in male but not female rats, activate liver Stat 5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation of this latent cytoplasmic transcription factor (Waxman, D. J., Ram, P. A., Park, S. H., and Choi, H. K. (1995) J. Biol. Chem. 270, 13262-13270). We demonstrate that physiological levels of GH can also activate Stat 1 and Stat 3 in liver tissue, but with a dependence on the dose of GH and its temporal plasma profile that is distinct from Stat 5 and with a striking desensitization following a single hormone pulse that is not observed with liver Stat 5. GH activation of the two groups of Stats leads to their selective binding to DNA response elements upstream of the c-fos gene (c-sis-inducible enhancer element; Stat 1 and Stat 3 binding) and the beta-casein gene (mammary gland factor element; liver Stat 5 binding). In addition to tyrosine phosphorylation, GH is shown to stimulate phosphorylation of these Stats on serine or threonine in a manner that either enhances (Stat 1 and Stat 3) or substantially alters (liver Stat 5) the binding of each Stat to its cognate DNA response element. These findings establish the occurrence of multiple, Stat-dependent GH signaling pathways in liver cells that can target distinct genes and thereby contribute to the diverse effects that GH and its sexually dimorphic plasma profile have on liver gene expression.

Growth hormone regulation of male-specific rat liver P450s 2A2 and 3A2: induction by intermittent growth hormone pulses in male but not female rats rendered growth hormone deficient by neonatal monosodium glutamate.

Growth hormone (GH) secretory patterns regulate the expression of several sex-dependent liver cytochrome P450 (CYP) genes. Studies using the hypophysectomized rat model have established that the intermittent plasma GH secretory pattern associated with adult male rats markedly stimulates liver expression of the male-specific CYP 2C11, a testosterone 2 alpha- and 16 alpha-hydroxylase, but is not required for expression of other male-specific liver enzymes, including CYP 2A2, a testosterone 15 alpha-hydroxylase, and CYP 3A2, a testosterone 6 beta-hydroxylase. In the present study, the effects of intermittent GH treatment on liver CYP expression were studied in adult rats rendered GH deficient by neonatal administration of monosodium glutamate (MSG), which depletes circulating adult GH without the global loss of other pituitary-dependent hormones that is associated with hypophysectomy. Restoration of the normal masculine circulating GH profile of six daily pulses (180-225 ng GH/ml/peak) in MSG-treated male rats by the use of an external pumping apparatus led to a substantial (30-50%) restoration of normal male levels of CYP 2A2 and CYP 3A2 activity, protein, and mRNA. GH pulsation at the nonphysiological frequencies of two or four times per day was less effective unless given at a dose that resulted in supraphysiological plasma GH levels. Although intermittent GH treatment can induce male-specific P450 expression in hypophysectomized female rats, the same hormone treatment did not stimulate CYP 2A2 or CYP 3A2 expression in MSG-treated female rats. Liver GH receptor mRNA levels at adulthood were not significantly altered by neonatal MSG treatment, suggesting that the unresponsiveness of MSG-treated females and the previously reported low responsiveness of MSG-treated males to GH-induced CYP 2C11 expression are not due to the absence of GH receptor. Moreover, normal liver IGF-1 mRNA levels were expressed in the MSG-treated female rats, suggesting that the liver GH receptor is functional in these animals. The present findings establish that the adult male-specific enzymes CYP 2A2 and CYP 3A2 can be positively regulated by intermittent GH pulsation despite their GH-independent expression in hypophysectomized rats. Moreover, neonatal MSG treatment, particularly in female rats, may lead to the loss of factors other than GH that are required for full expression of the pulsatile GH-stimulated CYP 2A2, 3A2, and 2C11 genes.

Intermittent plasma growth hormone triggers tyrosine phosphorylation and nuclear translocation of a liver-expressed, Stat 5-related DNA binding protein. Proposed role as an intracellular regulator of male-specific liver gene transcription.

Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription that are regulated by the temporal pattern of pituitary GH release, which is intermittent in male rats and nearly continuous in females. To investigate the influence of these GH secretory patterns on intracellular hepatocyte signaling, we compared the pattern of liver nuclear protein tyrosine phosphorylation in male and female rats. An M(r) approximately 93,000 polypeptide, p93, was found to be tyrosine phosphorylated to a high level in male but not female rats. GH, but not prolactin, rapidly stimulated p93 tyrosine phosphorylation in hypophysectomized rats. Intermittent plasma GH pulses triggered repeated p93 phosphorylation, while continuous GH exposure led to desensitization and a dramatic decline in liver nuclear p93. p93 was cross-reactive with two monoclonal antibodies raised to mammary Stat 5, whose tyrosine phosphorylation is stimulated by prolactin. Intermittent GH pulsation translocated liver Stat 5/p93 protein from the cytosol to the nucleus and also activated its DNA binding activity, as demonstrated using a mammary Stat 5-binding DNA element derived from the beta-casein gene. p93 is thus a liver-expressed, Stat 5-related DNA binding protein that undergoes tyrosine phosphorylation and nuclear translocation in response to intermittent plasma GH stimulation and is proposed to be an intracellular mediator of the stimulatory effects of GH pulses on male-specific liver gene expression.

Dehydroepiandrosterone 3 beta-sulphate is an endogenous activator of the peroxisome-proliferation pathway: induction of cytochrome P-450 4A and acyl-CoA oxidase mRNAs in primary rat hepatocyte culture and inhibitory effects of Ca(2+)-channel blockers.

The role of steroids related to the adrenal androgen dehydroepiandrosterone (5-androstene-3 beta-ol-17-one; DHEA) in regulating the expression of peroxisomal and cytochrome P-450 4A (CYP4A) enzymes active in fatty acid metabolism was assessed using a primary rat hepatocyte culture system. Exposure of hepatocytes to the peroxisome proliferator, clofibric acid (10-250 microM), for 48-96 h led to substantial increases in CYP4A protein, CYP4A1, CYP4A2 and CYP4A3 mRNAs, and the mRNAs encoding both forms of peroxisomal acyl-CoA oxidase (ACOX-I and ACOX-II), as judged by Northern-blot analysis using gene-specific oligonucleotide probes. Although DHEA treatment in vivo is effective in inducing these mRNAs in rat liver, it had no effect in the cultured hepatocytes. In contrast, treatment of the cells with DHEA 3 beta-sulphate (DHEA-S; 10-250 microM) stimulated major increases in CYP4A and ACOX mRNA levels. Examination of several analogues indicated a preference for 3 beta-sulphate over 17 beta-sulphated steroids and the inactivity of a 3 alpha-hydroxy-17 beta-sulphate derivative (DHEA-S > 5-androstene-3 beta,17 beta-diol 3-sulphate approximately 5 alpha-androstene-3 beta-ol-17-one 3-sulphate > 5-androstene-3 beta, 17 beta,17 beta-diol 17-sulphate approximately 5 beta-androstane-3 alpha-ol-17-one 3-sulphate >> 5 alpha-androstane-3 alpha, 17 beta-diol 17-sulphate). Induction of CYP4A mRNAs by either DHEA-S or clofibric acid was partially blocked by structurally diverse Ca(2+)-channel antagonists (nicardipine, nifedipine and diltiazem; 50 microM), suggesting that both the steroidal and fibrate classes of CYP4A inducers stimulate peroxisomal-proliferative responses via a Ca(2+)-dependent pathway. Retinoic acid alone slightly induced CYP4A mRNAs but did not enhance the induction by clofibrate or DHEA-S. As DHEA-S corresponds to a physiologically important major circulating androgen, these findings suggest that it may serve as an endogenous regulator of hepatic peroxisome enzyme levels. They further suggest that Ca(2+)-channel blockers may be useful pharmacological tools for the further study of the underlying cellular mechanism whereby endogenous steroids and fibrate drugs induce peroxisome proliferation, and the relationship of these events to activation of the peroxisome proliferator-activated receptor.

Irreversible suppression of growth hormone-dependent cytochrome P450 2C11 in adult rats neonatally treated with monosodium glutamate.

Neonatal exposure to monosodium glutamate (MSG) permanently blocks growth hormone (GH) secretion, which results in the development of a well-defined syndrome characterized by stunted body growth, obesity and impaired drug metabolism. We have found that restoration of the normal masculine circulating profile of GH (i.e., six daily pulses) by use of an external pumping apparatus is ineffective in restoring the normal expression of hepatic cytochrome P450 2C11, a major GH-dependent drug and steroid metabolizing enzyme that is eliminated by MSG treatment. Moreover, administering GH at two, four or seven plasma pulses per day with amplitudes ranging from physiologic to 7 times normal were similarly ineffective in restoring the expression (at both an activity and mRNA level) of the cytochrome. Additionally, multicytochrome P450-dependent hexobarbital hydroxylase was also unresponsive to GH administration in the MSG-treated rats. Because GH replacement was unable to correct the enzyme defects, our results suggest that the developmental abnormalities produced by neonatal MSG are not simply a result of a GH deficiency per se, but are due to an irreversible insensitivity of the target cell to GH.

Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms.

The role of thyroid hormone in regulating the expression of the flavoprotein NADPH cytochrome P450 reductase was studied in adult rats. Depletion of circulating thyroid hormone by hypophysectomy, or more selectively, by treatment with the anti-thyroid drug methimazole led to a 75-85% depletion of hepatic microsomal P450 reductase activity and protein in both male and female rats. Thyroxine substantially restored P450 reductase activity at a dose that rendered the thyroid-depleted rats euthyroid. Microsomal P450 reductase activity in several extrahepatic tissues was also dependent on thyroid hormone, but to a lesser extent than in liver (30-50% decrease in kidney, adrenal, lung, and heart but not in testis from hypothyroid rats). Hepatic P450 reductase mRNA levels were also decreased in the hypothyroid state, indicating that the loss of P450 reductase activity is not a consequence of the associated decreased availability of the FMN and FAD cofactors of P450 reductase. Parallel analysis of S14 mRNA, which has been studied extensively as a model thyroid-regulated liver gene product, indicated that P450 reductase and S14 mRNA respond similarly to these changes in thyroid state. In contrast, while the expression of S14 and several other thyroid hormone-dependent hepatic mRNAs is stimulated by feeding a high carbohydrate, fat-free diet, hepatic P450 reductase expression was not increased by this lipogenic diet. Injection of hypothyroid rats with T3 at a supraphysiologic, receptor-saturating dose stimulated a major induction of hepatic P450 reductase mRNA that was detectable 4 h after the T3 injection, and peaked at approximately 650% of euthyroid levels by 12 h. However, this same treatment stimulated a biphasic increase in P450 reductase protein and activity that required 3 days to reach normal euthyroid levels. T3 treatment of euthyroid rats also stimulated a major induction of P450 reductase mRNA that was maximal (12-fold increase) by 12 h, but in this case no major increase in P450 reductase protein or activity was detectable over a 3-day period. Together, these studies establish that thyroid hormone regulates P450 reductase expression by pretranslational mechanisms. They also suggest that other regulatory mechanisms, which may involve changes in P450 reductase protein stability and/or changes in the translational efficiency of its mRNA, are likely to occur.

Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450.

Plasma growth hormone (GH) profiles are sexually differentiated in many species and regulate the sex-dependence of peripubescent growth rates and liver function, including steroid hydroxylase cytochrome P450 expression, by mechanisms that are poorly understood. By use of an external pump to deliver to hypophysectomized rats pulses of rat GH of varying frequency and amplitude, a critical element for liver discrimination between male and female GH patterns was identified. Liver expression of the male-specific steroid 2 alpha (or 16 alpha)-hydroxylase P450, designated CYP2C11, was stimulated by GH at both physiological and nonphysiological pulse amplitudes, durations, and frequencies, provided that an interpulse interval of no detectable GH was maintained for at least 2.5 hr. This finding suggests that hepatocytes undergo an obligatory recovery period after stimulation by a GH pulse. This period may be required to reset a GH-activated intracellular signaling pathway or may relate to the short-term absence of GH receptors at the hepatocyte surface after a cycle of GH binding and receptor internalization. These requirements were distinguished from those necessary for the stimulation by GH of normal male growth rates in hypophysectomized rats, indicating that different GH responses and, perhaps, different GH-responsive tissues recognize distinct signaling elements in the sexually dimorphic patterns of circulating GH.

Hepatic P450 expression in hypothyroid rats: differential responsiveness of male-specific P450 forms 2a (IIIA2), 2c (IIC11), and RLM2 (IIA2) to thyroid hormone.

Studies carried out in hypophysectomized adult rats have demonstrated that both thyroid hormone and GH can suppress hepatic expression of the steroid 6 beta-hydroxylase P450 2a (IIIA2). The present study further characterizes the influence of thyroid hormone on the expression of P450 2a and two other male-specific hepatic P450s, a steroid 2 alpha/16 alpha-hydroxylase, designated P450 2c (IIC11), and a steroid 15 alpha-hydroxylase, designated P450 RLM2 (IIA2). These studies were carried out in rats rendered hypothyroid by treatment with methimazole, which allows for the nonsurgical depletion of circulating T4, and in hypophysectomized rats. Hypothyroidism led to an increase in hepatic P450 2a (IIIA2) protein and mRNA in both male and female rats that was fully reversed by T4 replacement. In contrast, hypothyroidism decreased by 70-80% the expression of P450 2c (IIC11) activity and mRNA, but did not significantly alter the expression of P450 RLM2 (IIA2). The decrease in P450 2c (IIC11) was not reversed by T4 replacement, suggesting that it is a consequence of the loss of plasma GH pulses that occurs secondary to hypothyroidism. In agreement with these findings, T4 given to hypophysectomized rats partially suppressed the expression of P450 2a (IIIA2) mRNA, but not P450 2c (IIC11) or P450 RLM2 (IIA2) mRNA. A more complete suppression of P450 2a (IIIA2) mRNA as well as P450 2c (IIC11) mRNA was achieved when the hypophysectomized rats were treated with T3 at a supraphysiological, receptor-saturating dose. Although GH administered to intact male rats by continuous infusion fully suppressed all three male-specific P450 proteins and their mRNAs, the same treatment given to hypothyroid rats was only partially suppressive in the case of P450 2a (IIIA2) and P450 RLM2 (IIA2), unless combined with T4. In the case of P450 2c (IIC11), substantial suppression of the residual P450 present in hypothyroid rats was achieved by treatment with GH alone, despite persistent thyroid hormone deficiency. These studies demonstrate that while thyroid hormone is a negative regulator of P450 2a (IIIA2) expression and is required for the full suppression of that P450 and P450 RLM2 (IIA2) by the continuous plasma GH profiles associated with adult female rats, the suppression of P450 2c (IIC11) by continuous plasma GH is largely independent of the presence of thyroid hormone.

Pretranslational control by thyroid hormone of rat liver steroid 5 alpha-reductase and comparison to the thyroid dependence of two growth hormone-regulated CYP2C mRNAs.

The sexually differentiated microsomal enzyme steroid 5 alpha-reductase (NADPH: delta 4-3-oxosteroid 5 alpha-oxido-reductase, EC 1.3.99.5) catalyzes the NADPH-dependent conversion of testosterone to 5 alpha-dihydrotestosterone, a more potent androgen. In rat liver, this enzyme is expressed at a 10-fold higher level in adult females as compared to adult males. The pituitary regulation of this enzyme and its mRNA was studied in untreated and hypophysectomized rats and in rats rendered hypothyroid by treatment with the antithyroid drug methimazole. Hepatic 5 alpha-reductase activity was elevated 8-fold, to 85% of adult female levels, in adult male rats given growth hormone by continuous infusion. This same treatment was only partially effective in restoring 5 alpha-reductase in rats depleted of endogenous growth hormone by hypophysectomy, indicating that other pituitary-dependent factors contribute to the elevation observed in the inact animals. Further analysis revealed that thyroxine, but not adrenocorticotropic hormone (ACTH) or chorionic gonadotropin, could elevate 5 alpha-reductase activity and mRNA when given to the hypophysectomized rats and that this effect was enhanced by the presence of growth hormone. This thyroid hormone dependence was confirmed by the decrease in hepatic 5 alpha-reductase expression in hypothyroid rats and by its substantial restoration following thyroxine replacement. Thyroxine also stimulated expression of another female-predominant hepatic mRNA, encoding the steroid 16 alpha-hydroxylase cytochrome P-450f (IIC7), in a manner that was independent of the stimulatory effect of growth hormone on this transcript. In contrast, thyroid hormone did not significantly affect protein or mRNA levels of the growth hormone-stimulated, female-specific steroid sulfate 15 beta-hydroxylase P-450 2d (IIC12). These findings establish that thyroid hormones act at a pretranslational level to modulate the expression of some, but not all, growth hormone-stimulated hepatic mRNAs and demonstrate that both thyroxine and growth hormone can independently contribute to the sex-dependent expression of hepatic enzymes of steroid metabolism.

Pituitary regulation of the male-specific steroid 6 beta-hydroxylase P-450 2a (gene product IIIA2) in adult rat liver. Suppressive influence of growth hormone and thyroxine acting at a pretranslational leve;.

Oligonucleotide probes that distinguish between two closely related mRNAs encoding steroid 6 beta-hydroxylases of rat P-450 gene family CYP3A were used to individually assess their responsiveness to pituitary hormone regulation. Northern blot analysis revealed that the elevation of immunoreactive P-450 IIIA2 in livers of hypophysectomized rats reflects an elevation of the constitutive, male-specific P-450 IIIA2 (P-450 2a) and not an induction of the drug-inducible P-450 IIIA1 (P-450p). P-450 IIIA2 mRNA levels in intact adult male rats were found to be markedly reduced by GH administered as a continuous infusion at levels as low as 1 mU/h, indicating that GH acts at a pretranslational step to suppress expression of this P-450 enzyme. In hypophysectomized male rats, however, this same hormone treatment was only partially effective at suppressing P-450 IIIA2 mRNA and protein, suggesting that other pituitary-dependent factors contribute to the suppression observed in the intact rats. Further analysis revealed that T4, but not ACTH or human CG, can act in concert with GH to effect a more complete suppression of hepatic P-450 IIIA2 mRNA and protein in hypophysectomized rats. T4 also suppressed the expression of another GH-regulated, male-specific hepatic enzyme, designated P-450 IIA2 (P-450 RLM2), particularly in hypophysectomized female rats. In contrast, the GH-responsive P-450 IIA1 (P-450 3) was much less affected by T4 treatment. Thus, while T4 can modulate P-450 IIIA2 expression, it does not serve as a universal regulator for hepatic expression of GH-responsive P-450s.

Thin-layer chromatographic method for the determination of glycosyltransferase activity.

To survey glycosyltransferase activities and specificities we have developed a TLC method to separate various nucleotide sugars from both high- and low-molecular-weight sugar acceptors. Here, we report details of the procedure and its application for galactosyltransferase and fucosyltransferase detected in mouse spermatogenic cells. The assay method involves sample separation using polyethyleneimine cellulose plastic-backed thin-layer plates, developed in sodium phosphate buffer for 30 min. Nucleotide sugars, including UDP-Gal, GDP-Fuc, CMP-NeuNAc, and GDP-Man, remain at the origin, while both high- and low-molecular-weight sugar acceptors migrate within 2 cm of the solvent front. Assays for galactosyltransferase and fucosyltransferase are linear with time and yield results comparable to other methods such as gel permeation chromatography and micropartitioning filtration. The TLC protocol should be useful for determinations of many different glycosyltransferases.

Expression and topographical localization of cell surface fucosyltransferase activity during epididymal sperm maturation in the mouse.

We have demonstrated previously that spermatogenic cells in the mouse testis have high levels of fucosyltransferase activity. Furthermore, a significant portion of this activity has been localized to the cell surface (Millette et al.: Cell Biology of the Testis and Epididymis, 1987). Differential expression of fucosyltransferases and their function as ecto-enzymes may be important in the processes of sperm maturation and fertilization in mammals. Accordingly, here we report the activity levels of fucosyltransferase (FT) in spermatozoa isolated from the mouse caput and cauda epididymides. Calculated on a per cell basis, spermatozoa from the caput epididymis have significantly more FT activity than do spermatozoa from the cauda epididymis (18.07 +/- 2.2 pmol/million cells compared with 2.8 +/- 0.09 pmol/million cells). Furthermore, caput sperm exhibit a more significant increase in FT activity when assayed in the presence of Nonidet P-40. Calculated on the basis of cell surface area, however, FT activity remains constant on the head portion of spermatozoa isolated from all portions of the male reproductive tract and from capacitated spermatozoa. Measurements of FT activity in extracts of isolated sperm tails from cells at different stages of maturation indicate a greatly diminished activity in tails from sperm in the cauda epididymis. The total sperm surface area is composed predominantly of the plasma membrane surrounding the flagellar apparatus. Therefore, our data demonstrate that FT activity is retained selectively on the different topological regions of sperm, with losses during sperm maturation in the epididymis being restricted to the tail segment. Maintenance of high levels of FT activity of the plasma membranes of the mouse sperm head raise the possibility that FT is indeed involved in some aspects of sperm-egg recognition.