Helen W. Davey

Distinctive Roles of STAT5a and STAT5b in Sexual Dimorphism of Hepatic P450 Gene Expression IMPACT OF Stat5a GENE DISRUPTION

Stat5b gene disruption leads to an apparent growth hormone (GH) pulse insensitivity associated with loss of male-characteristic body growth rates and male-specific liver gene expression (Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7239–7244). In the present study, disruption of the mouse Stat5a gene, whose coding sequence is ∼90% identical to the Stat5b gene, resulted in no loss of expression in male mice of several sex-dependent, GH-regulated liver cytochrome P450 (CYP) enzymes. By contrast, the loss of STAT5b feminized the livers of males by decreasing expression of male-specific CYPs (CYP2D9 and testosterone 16α-hydroxylase) while increasing to female levels several female-predominant liver CYPs (CYP3A, CYP2B, and testosterone 6β-hydroxylase). Since STAT5a is thus nonessential for these male GH responses, STAT5b homodimers, but not STAT5a-STAT5b heterodimers, probably mediate the sexually dimorphic effects of male GH pulses on liver CYP expression. In female mice, however, disruption of either Stat5a or Stat5bled to striking decreases in several liver CYP-catalyzed testosterone hydroxylase activities. Stat5a or Stat5b gene disruption also led to the loss of a female-specific, GH-regulated hepatic CYP2B enzyme. STAT5a, which is much less abundant in liver than STAT5b, and STAT5b are therefore both required for constitutive expression in female but not male mouse liver of certain GH-regulated CYP steroid hydroxylases, suggesting that STAT5 protein heterodimerization is an important determinant of the sex-dependent and gene-specific effects that GH has on the liver.

STAT5b mediates the GH-induced expression of SOCS-2 and SOCS-3 mRNA in the liver.

Suppressor of cytokine signalling (SOCS) proteins act as part of a classical negative feedback loop regulating cytokine signal transduction. Expression of SOCS proteins is induced in response to cytokines and down-regulates the cytokine signal by inhibiting the JAK/STAT pathway. Growth hormone (GH) was previously shown to induce strong transient expression of SOCS-3 and to a lesser extent CIS, SOCS-1 and SOCS-2 in mouse liver (Adams, T.E., Hansen, J.A., Starr, R., Nicola, N.A., Hilton, D.J., Billestrup, N., 1998. Growth hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signalling. J. Biol. Chem. 273, 1285-1287.). In this work we have compared GH-induced SOCS gene expression in wild-type and STAT5b-deficient mice, and show that STAT5b is required for the induction of SOCS-2 and SOCS-3 in liver. In contrast, the absence of STAT5b has no effect on the GH-induced expression of CIS and SOCS-2 mRNA in the mammary gland. Suprisingly, there is no activation of SOCS-3 expression in mammary glands of wild-type and STAT5b mutant mice following GH administration. These results highlight both tissue- and factor-specific differences in the regulation of SOCS gene expression by STAT5a/b.

STAT5b-deficient Mice Are Growth Hormone Pulse-resistant ROLE OF STAT5b IN SEX-SPECIFIC LIVER P450 EXPRESSION

The signal transducer and transcriptional activator STAT5b is required to maintain the adult male pattern of liver gene expression and whole body pubertal growth rates, as demonstrated by the loss of these growth hormone (GH) pulse-dependent responses in mice with a targeted disruption of the STAT5b gene. The present study investigates whether these phenotypes of STAT5b-deficient mice result from impaired intracellular GH signaling associated with a loss of GH pulse responsiveness, as contrasted with a feminization of the pituitary GH secretory profile leading to the observed feminization of body growth and liver gene expression. Pulsatile GH replacement in hypophysectomized mice stimulated body weight gain in wild-type but not in STAT5b-deficient mice. Expression of the male-specific liver P450 enzyme CYP2D9, which is reduced to female levels in hypophysectomized male mice, was restored to male levels by GH pulse replacement in wild-type but not in STAT5b-deficient mice. Similarly, a female-specific liver CYP2B P450 enzyme that was up-regulated to female levels following hypophysectomy of males was suppressed to normal basal male levels by GH pulses only in wild-type hypophysectomized mice. Finally, urinary excretion of the male-specific, GH pulse-induced major urinary protein was restored to normal male levels following pulsatile GH treatment only in the case of wild-type hypophysectomized mice. STAT5b-deficient mice are thus GH pulse-resistant, supporting the proposed role of STAT5b as a key intracellular mediator of the stimulatory effects of plasma GH pulses on the male pattern of liver gene expression.

Ontogeny and epithelial-stromal interactions regulate IGF expression in the ovine mammary gland.

Although the insulin-like growth factors (IGF-I and -II) have been implicated in the stimulation of mammogenesis, little is known of their regulation in the mammary gland. In this study we removed epithelial tissue from one of the two mammary glands of 1-week-old ewe lambs and examined IGF-I and -II mRNA expression during postnatal development in both the intact mammary gland and in the gland cleared of epithelial tissue. Expression of IGF-I mRNA was highest at 6 and 10 weeks of age, coincident with the prepubertal phase of rapid mammary growth, then declined and remained low until expression increased during late pregnancy. IGF-I mRNA was more abundant in the mammary fat pad adjacent to parenchyma (MFP) than in the contralateral fat pad that had been surgically cleared of epithelium (CFP). The level of IGF-II mRNA in parenchyma was highest at 1-23 weeks of age due to an increase in the abundance of specific mRNAs. Expression was lower in the fat pads, with generally higher levels in the intact MFP than the CFP, and in these tissues IGF-II expression was shown to increase with age between 6 and 23 weeks. We also investigated the influence of the ovary and estrogen on the expression of IGFs. While IGF-I mRNA abundance was unaffected by ovariectomy, exogenous estrogen resulted in higher levels of expression in the MFP of ovariectomized ewes and tended to increase its level in the parenchyma of intact ewes. Ovariectomy increased IGF-II mRNA within mammary parenchyma whereas estrogen suppressed levels in both the parenchyma and MFP. These findings demonstrate that IGF-I and -II mRNAs are expressed locally within the developing ovine mammary gland and are regulated by stage of ontogeny, ovarian hormones, and epithelial stromal interaction.

Essential Role of Stat5 for IL-5-Dependent IgH Switch Recombination in Mouse B Cells

IL-5 stimulation of CD38-activated murine splenic B cells induces μ-γ1 CSR at the DNA level leading to a high level of IgG1 production. Further addition of IL-4 in the system enhances IL-5-dependent μ-γ1 CSR. Although some of the postreceptor signaling events initiated by IL-5 in activated B cells have been characterized, the involvement of Stat in IL-5 signaling has not been thoroughly evaluated. In this study, we examined the activation of Stat5 and activation-induced cytidine deaminase (AID) in CD38-activated murine splenic B cells by IL-5. The role of Stat5a and Stat5b in IL-5-induced μ-γ1 CSR and also IgG1 and IgM production was documented, as IL-5 does not act on CD38-stimulated splenic B cells from Stat5a−/− and Stat5b−/− mice. Expression levels of CD38-induced germline γ1 transcripts and AID in Stat5a−/− and Stat5b−/− B cells upon IL-5 stimulation were comparable to those of wild-type B cells. The impaired μ-γ1 CSR by Stat5b−/− B cells, but not by Stat5a−/− B cells, was rescued in part by IL-4, as the addition of IL-4 to the culture of CD38- and IL-5-stimulated B cells induced μ-γ1 CSR leading to IgG1 production. Analysis of cell division cycle number of wild-type B cells revealed that μ-γ1 CSR was observed after five or six cell divisions. Stat5a−/− and Stat5b−/− B cells showed similar cell division cycles, but they did not undergo μ-γ1 CSR. Our data support the notion that both Stat5a and Stat5b are essential for IL-5-dependent μ-γ1 CSR and Ig secretion; however, their major target may not be AID. Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.

The sheep (Ovis aries) H19 gene: genomic structure and expression patterns, from the preimplantation embryo to adulthood

H19, which is one of the most abundantly expressed imprinted genes during mammalian embryonic and foetal development, has been cloned from a ruminant. The sheep (Ovis aries) gene contains five exons interspersed by four exceptionally small introns; only short stretches of the nucleotide sequence, particularly in exon 1, show good homology with the human gene. The size of the exons and introns and the sequences around the splice junctions however, are well conserved between the species. The gene encodes a approximately 2.6 kb transcript which contains several potential short open reading frames, none of which is conserved between the ovine and human or murine transcripts, supporting a previous hypothesis that the gene product is the untranslated RNA itself. H19 mRNA is highly abundant in most ovine embryonic and foetal tissues of mesodermal and endodermal origins but was not detected in tissues of ectodermal origin such as the trophectoderm and the foetal brain. Expression of H19 in the extraembryonic membranes was detected only after the ovine conceptus began attachment to the endometrium and the embryo itself had undergone early organogenesis. This may be regarded as the first step in implantation; thus, in comparison with the mouse, the initiation of H19 expression appears to be determined by the timing of implantation rather than by the stage of development of the embryo itself. In most tissues, H19 expression is temporally linked to IGF2, a major foetal growth factor. The exceptions were the elongated blastocyst, the trophectoderm and brain, where low levels of IGF2 were observed in the absence of detectable H19. The abundance of H19 mRNA was in general, directly correlated with IGF2 mRNA abundance in mesodermal and endodermal tissues, suggesting that the two ovine genes share common regulatory elements that co-ordinately regulate their expression. Though both are generally regarded as embryonic and foetal genes, their expression was still maintained at a fairly high level in the adult sheep liver, lung, skeletal muscle, adrenal gland and kidney, suggesting that these organs are significant sources of IGF II in the adult.

Paracrine action of keratinocyte growth factor (KGF) during ruminant mammogenesis.

Keratinocyte growth factor (KGF) is a stroma-derived mitogen mediating epithelial-stromal interactions. We investigated the role of KGF during epithelial-stromal interactions accompanying ruminant mammogenesis. Target-specificity of KGF was demonstrated in that KGF-stimulated proliferation of bovine mammary epithelial, but not ovine mammary stromal cells. Consistent with a paracrine function, 4.6, 2.4, 1.5 and 0.9 kb mRNA transcripts were expressed by bovine stromal, but not epithelial cells. Within the ovine mammary gland, 2.4 and 1.5 kb KGF mRNAs were expressed in the fat pad while only the 2.4 kb transcript was transcribed in parenchyma. The abundance of KGF mRNA was greater in the extra-parenchymal fat pad than in the contralateral epithelium-free fat pad prior to puberty, and was less in parenchyma than in the intact or epithelium-free fat pads. Ovariectomy tended to increase KGF transcription while estrogen reduced expression. Of several tissues, mammary parenchyma expressed a 2.4 kb mRNA while adipose tissues expressed a 1.5 kb transcript. These results demonstrate local and systemic regulation of KGF transcription and support a paracrine role for KGF during ruminant mammogenesis.