Sexual Dimorphism of Rat Liver Nuclear Proteins

Many genes are expressed in mammalian liver in a sexually dimorphic manner. DNA microarray analysis has shown that growth hormone (GH) and its sex-dependent pattern of pituitary secretion play a major role in establishing the sexually dimorphic patterns of liver gene expression. However, GH may exert effects on protein post-translational modification and nuclear localization that are not reflected at the mRNA level. To investigate these potential effects of GH, we used two-dimensional gel electrophoresis followed by LC-MS/MS to: 1) identify rat liver nuclear proteins whose abundance or state of post-translational modification displays sex-dependent differences; and 2) determine the role of the plasma GH profile in establishing these differences. Nuclear extracts prepared from livers of individual male (n = 9) and female (n = 5) adult rats, and from males given GH by continuous infusion for 7 days to feminize liver gene expression (n = 5 rats), were resolved by two-dimensional electrophoresis. Image analysis of SYPRO Ruby-stained gels revealed 165 sexually dimorphic protein spots that differ in normalized volume between male and female groups by >1.5-fold at p < 0.05. Sixty of these proteins exhibited female-like changes in spot abundance following continuous GH treatment. Comparison of male and GH-treated male groups revealed 130 proteins that displayed >1.5-fold differences in abundance, with 60 of these GH-responsive spots being sexually dimorphic. Thus, GH plays an important role in establishing the sex-dependent differences in liver nuclear protein content. Twenty-eight of the sexually dimorphic and/or GH-regulated protein spots were identified by LC-MS/MS. Proteins identified include regucalcin, nuclear factor 45, and heterogeneous nuclear ribonucleoproteins A3, D-like, and K, in addition to proteins such as GST, normally associated with cytosolic extracts but also reported to be localized in the nucleus. Many genes are expressed in mammalian liver in a sexually dimorphic manner. DNA microarray analysis has shown that growth hormone (GH) and its sex-dependent pattern of pituitary secretion play a major role in establishing the sexually dimorphic patterns of liver gene expression. However, GH may exert effects on protein post-translational modification and nuclear localization that are not reflected at the mRNA level. To investigate these potential effects of GH, we used two-dimensional gel electrophoresis followed by LC-MS/MS to: 1) identify rat liver nuclear proteins whose abundance or state of post-translational modification displays sex-dependent differences; and 2) determine the role of the plasma GH profile in establishing these differences. Nuclear extracts prepared from livers of individual male (n = 9) and female (n = 5) adult rats, and from males given GH by continuous infusion for 7 days to feminize liver gene expression (n = 5 rats), were resolved by two-dimensional electrophoresis. Image analysis of SYPRO Ruby-stained gels revealed 165 sexually dimorphic protein spots that differ in normalized volume between male and female groups by >1.5-fold at p < 0.05. Sixty of these proteins exhibited female-like changes in spot abundance following continuous GH treatment. Comparison of male and GH-treated male groups revealed 130 proteins that displayed >1.5-fold differences in abundance, with 60 of these GH-responsive spots being sexually dimorphic. Thus, GH plays an important role in establishing the sex-dependent differences in liver nuclear protein content. Twenty-eight of the sexually dimorphic and/or GH-regulated protein spots were identified by LC-MS/MS. Proteins identified include regucalcin, nuclear factor 45, and heterogeneous nuclear ribonucleoproteins A3, D-like, and K, in addition to proteins such as GST, normally associated with cytosolic extracts but also reported to be localized in the nucleus. Growth hormone (GH), 1The abbreviations used are: GH, growth hormone; 2D, two-dimensional; STAT5, signal transducer and activator of transcription 5; hnRNP, heterogeneous nuclear ribonucleoprotein; NF45, nuclear factor 45. 1The abbreviations used are: GH, growth hormone; 2D, two-dimensional; STAT5, signal transducer and activator of transcription 5; hnRNP, heterogeneous nuclear ribonucleoprotein; NF45, nuclear factor 45. a polypeptide hormone secreted by the anterior lobe of the pituitary gland, plays a major role in the regulation of metabolism and longitudinal growth. Primary targets of GH action include the liver, skeletal muscle, and adipose tissue. In many species, including humans, the temporal pattern of pituitary GH secretion differs between males and females, resulting in sexually dimorphic plasma GH profiles (1Jansson J.O. Eden S. Isaksson O. Sexual dimorphism in the control of growth hormone secretion..Endocr. Rev. 1985; 6: 128-150Google Scholar, 2Veldhuis J.D. Gender differences in secretory activity of the human somatotropic (growth hormone) axis..Eur. J. Endocrinol. 1996; 134: 287-295Google Scholar, 3Winer L.M. Shaw M.A. Baumann G. Basal plasma growth hormone levels in man: New evidence for rhythmicity of growth hormone secretion..J. Clin. Endocrinol. Metab. 1990; 70: 1678-1686Google Scholar). Sex differences in plasma GH profiles are especially prominent in rodents. In adult male rats, GH is released into circulation every 3–4 h, which gives rise to high plasma GH peaks separated by periods of very low to undetectable plasma GH (4Tannenbaum G.S. Martin J.B. Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat..Endocrinology. 1976; 98: 562-570Google Scholar). In contrast, adult female rats are characterized by lower yet more frequent, overlapping pulses of GH secretion, resulting in the presence of GH in plasma at nearly all times (5Eden S. Age- and sex-related differences in episodic growth hormone secretion in the rat..Endocrinology. 1979; 105: 555-560Google Scholar, 6Painson J.C. Tannenbaum G.S. Sexual dimorphism of somatostatin and growth hormone-releasing factor signaling in the control of pulsatile growth hormone secretion in the rat..Endocrinology. 1991; 128: 2858-2866Google Scholar). GH acts via these sex-dependent plasma profiles to establish sex differences in body growth rates (7Jansson J.O. Albertsson-Wikland K. Eden S. Thorngren K.G. Isaksson O. Effect of frequency of growth hormone administration on longitudinal bone growth and body weight in hypophysectomized rats..Acta Physiol. Scand. 1982; 114: 261-265Google Scholar) and in the sexually dimorphic expression of a large number of genes in the liver. These genes encode various receptors, signal transduction molecules, and enzymes of steroid and foreign compound metabolism, in particular cytochrome P450 (8Sundseth S.S. Alberta J.A. Waxman D.J. Sex-specific, growth hormone-regulated transcription of the cytochrome P450 2C11 and 2C12 genes..J. Biol. Chem. 1992; 267: 3907-3914Google Scholar, 9Mode A. Tollet P. Strom A. Legraverend C. Liddle C. Gustafsson J.A. Growth hormone regulation of hepatic cytochrome P450 expression in the rat..Adv. Enzyme Regul. 1992; 32: 255-263Google Scholar, 10Legraverend C. Mode A. Westin S. Strom A. Eguchi H. Zaphiropoulos P.G. Gustafsson J.A. Transcriptional regulation of rat P-450 2C gene subfamily members by the sexually dimorphic pattern of growth hormone secretion..Mol. Endocrinol. 1992; 6: 259-266Google Scholar, 11Srivastava P.K. Waxman D.J. Sex-dependent expression and growth hormone regulation of class alpha and class mu glutathione S-transferase mRNAs in adult rat liver..Biochem. J. 1993; 294: 159-165Google Scholar, 12Ahluwalia A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar). DNA microarray analysis has shown that GH a major of sexually dimorphic liver gene expression in the rat with GH the expression of of genes to sex differences in expression A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar). However, changes in gene expression at the mRNA not to changes at the protein level. GH may post-translational changes that and protein patterns via signal transduction that are not at the of gene expression and not be by microarray gel electrophoresis with is a to the state of protein expression in and and its to including hormone used to identify changes in protein expression in including and C. J. S. P. analysis of human Scholar, H. in of Scholar, of liver Scholar, H. G. pattern of protein expression in from in to to A. A. J. in protein expression profiles in to Scholar) and H. S. two-dimensional and of nuclear proteins from as for of transcription J. and S. protein expression at the of in the Biol. Chem. Scholar, analysis of nuclear proteins of in Scholar, S. P. J. P. A. analysis of nuclear proteins from and human Scholar). In the nuclear extracts prepared from rat liver were by gel electrophoresis followed by LC-MS/MS in to identify proteins that sex-dependent differences in abundance and to determine the role of GH in establishing these differences. of 165 spots expressed between males and were on the and 60 of these spots exhibited female-like changes in abundance in to continuous GH treatment. Twenty-eight sex-dependent and/or GH-regulated spots were including proteins that may to GH signaling and in the nucleus. male and female rats were from rats were with GH by continuous an as D.J. plasma growth hormone and nuclear of a DNA role as an of liver gene Biol. Chem. Scholar). with GH were on the of male rats to GH at body for 7 the plasma GH profile of adult female rats and the liver mRNA profile A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar). nuclear extracts were prepared from individual rat livers to the by K. in transcription from the Scholar) and in as D.J. plasma growth hormone and nuclear of a DNA role as an of liver gene Biol. Chem. Scholar). Nuclear extracts prepared in are in transcription such as signal transducer and activator of transcription 5 and hepatic nuclear factor as by activity for individual liver nuclear by a DNA D.J. plasma growth hormone and nuclear of a DNA role as an of liver gene Biol. Chem. Scholar). nuclear extracts that exhibited low or activity with the in the were as nuclear extracts with low activity Waxman D.J. growth hormone of hepatic regulation and role in liver gene Scholar). from individual nuclear a and in 7 and a of of were separated by and an from a at at low for followed by 60 for the were to for h, followed by for h, for h, a to for h, and for at were at to in the gels and an from in of and a of followed by in of the were at on of the gel a at at gel for the followed by gel for were for in acid and in the at for at with gel of SYPRO were in acid for and and to spot the gels were in at SYPRO Ruby-stained gels were with a an of and an of were spots were by with from gel were to the spots on an by followed by with and of the gel on the of of the protein spots not on that gel were to the gel from the the spot by on gel were normalized as a of the volume of spots that were on all were from to and to for is for analysis of gene expression the spot for gel spots were into the as and individual normalized spot were as were to for all spots that were or whose normalized volume normalized were for spots of the groups males (n = (n = 5 and GH-treated males (n = 5 and between groups in a normalized spot were also for the of male nuclear males with high liver activity and males with low liver activity for the differences in normalized of spots between groups at groups were with to the of spot volume differences a with p < were to be were on the the by the were on the of low protein on spots to be identified were from the gels an the protein spot were from to gels and for LC-MS/MS analysis to the were to for at with of followed by with an LC-MS/MS on a with a of acid in in acid in a of in followed by of the were or In the of protein as heterogeneous nuclear were with the with or used the following a of for and and and as were to a that with a of and a of with for used to the and the state from the and were at a and a of or for the or were as and as and and for and nuclear extracts prepared from individual male and female rats were by gel electrophoresis to identify nuclear proteins whose abundance or state of post-translational modification displays sex-dependent differences. To determine the role of GH in establishing these sex we also liver nuclear extracts prepared from male rats given GH as a continuous the plasma GH levels that are of adult female rats and the expression of liver proteins in of to changes in spot abundance between liver SYPRO Ruby-stained gel of male rat liver nuclear is shown in and spots were resolved on Comparison of male and female liver nuclear revealed 165 spots that exhibited >1.5-fold differences in abundance at p < 0.05. of the 165 sexually dimorphic spots were in males with and spots were normalized volume in from to for these 165 Comparison with GH-treated male gels revealed that a of the sexually dimorphic of 165 spots to continuous GH in abundance between males and GH-treated Sixty of these sexually GH-regulated spots of the 165 sexually dimorphic exhibited female-like changes in abundance, with spots and spots by continuous GH treatment. of the 60 spots and the changes were at p < of female-like exhibited by these 60 sexually dimorphic protein spots GH of spots and GH of that the continuous GH is a factor that the sex-dependent expression of these proteins in rat liver. spots were and spots were by continuous of these changes sex-dependent pattern of expression of these protein and that of the and spots that not to continuous GH may be by of 165 nuclear protein spots sex-dependent patterns of expression in male rat to continuous GH of of of of < of < in a Comparison of male and continuous GH-treated male nuclear extracts revealed 130 protein spots that were to continuous in abundance between the at p < 0.05. of these spots were and were in the GH-treated males in spot abundance normalized volume from by to by Sixty of the 130 GH-responsive spots displayed sexually dimorphic expression of the 60 the to continuous GH in males the pattern of expression. Thus, spots by continuous GH were also in as with and spot in the GH-treated males were more in in GH plays a role in establishing sex-dependent differences in the nuclear protein of 130 nuclear protein spots to continuous GH in male rat in of of of < < by GH by GH in a of nuclear protein spots by normalized spot and the p for a are shown in and the spot are shown in spots for were on all male but be from number of the female the spots identified were on all female the spots were and 7 were in with sex-dependent differences in abundance the for all but protein spots and of the spots were to GH, as exhibited female-like changes in abundance in the continuous GH-treated males or of these the changes were of the spots were not to continuous GH of the proteins identified number of and and and is in and are shown in a of and GH-treated male with sexually dimorphic protein spots identified by by dimorphic rat liver nuclear proteins identified by spot volume spot volume GH spot volume p p dimorphic and GH-regulated acid acid dimorphic but not GH-regulated not the p be all normalized spot in and GH-treated an of not the p be all normalized spot in and GH-treated an of in a of nuclear proteins identified by or to rat of the protein in the identified of the were as a and acid acid following to the and following to the and following to the and but not following to the and protein or to rat of the protein in the of the were as a following to the and following to the and following to the and following to the and in a also to sexually GH-regulated protein spots whose abundance with the activity of the transcription factor major of in the plays a role in sexually dimorphic effects of GH on liver gene transcription Waxman D.J. of and in dimorphism of hepatic P450 gene expression. of gene Biol. Chem. Scholar). activity with the plasma GH levels in male rats, with nuclear activity high at the of plasma GH and activity low between GH pulses Waxman D.J. growth hormone of hepatic regulation and role in liver gene Scholar, G.S. Waxman D.J. between the sexually dimorphic GH secretory profiles and hepatic Scholar). To GH-regulated proteins whose nuclear abundance with the of a male GH the male gels were into males with high nuclear activity and males with low activity normalized were for protein spots and with in Comparison of the high and low groups revealed spots that exhibited differences in abundance between the groups at p < to high normalized spot volume = of these spots were and were in low males with high Comparison of the spots with the spots expressed at p < between and and between GH-treated males and males male and revealed spots in Comparison of the spots with spots that in abundance p < between and high and between GH-treated males and high also revealed spots in However, of the spots with spots that were or p < in and GH-treated males with low males revealed spots in spots were in the low males as with high females, and GH-treated of these spots not gel electrophoresis with LC-MS/MS to identify rat liver nuclear proteins whose abundance or state of post-translational modification sex-dependent and to determine the role of GH in establishing DNA microarray which that plasma GH profiles play a major role in establishing sex-dependent gene expression in rat liver A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar). Comparison of gel of nuclear extracts from males and revealed 165 protein spots that displayed a Thus, of the nuclear protein spots resolved on these gels were sexually dimorphic in Sixty of the 165 spots were GH-regulated and displayed female-like changes in expression following continuous GH of male is lower the of sex-dependent liver mRNAs that displayed GH regulation in microarray analysis in the rat liver of or A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar). gel from GH-treated males and male were 130 spots a to continuous GH were of the nuclear of these spots or displayed sex-dependent patterns of expression that with to the effects of continuous GH treatment. these that GH plays an important role in establishing sex-dependent patterns of rat liver nuclear is prominent that displayed by GH in establishing sexually dimorphic differences in liver mRNA expression A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar). of the sexually dimorphic protein spots were not to GH be in addition to GH, that to establishing and the sex-dependent patterns of liver nuclear proteins in the rat of the 130 GH-regulated nuclear proteins not that a of liver nuclear proteins be by GH in a that is of the sex-dependent effects that GH on liver gene expression. regulation may include GH signaling that not to changes in gene GH signaling to changes in protein by protein or protein D.J. Growth hormone via a of Scholar). of protein spots by of these spots to proteins with nuclear These include nuclear factor G. J. J. and expression of and nuclear factor of and Biol. Chem. acid C. A. and of the nuclear acid from rat Biol. Chem. 1992; 267: and H. of the of heterogeneous nuclear protein and its with Biol. Chem. K. J. nuclear A3, a Biol. Chem. and J. K. and expression of a that the in as the of the human heterogeneous nuclear of a Biol. Chem. all of which are nuclear in are to between the and the of mRNA Scholar). identified regucalcin, is in the and the H. of to rat liver of nuclear protein and protein J. 6: Scholar). Nuclear localization has also reported for cytosolic proteins identified in the S. of a acid protein from rat liver as A. 1985; Scholar, is to the of Biol. 1990; Scholar) and and protein is a glutathione S-transferase localized to of the Biol. and for which is in the G. in nuclear and of rat liver..Biochem. Scholar, Comparison of the of nuclear and from Scholar). a cytosolic has on gel of nuclear extracts from human H. S. two-dimensional and of nuclear proteins from as for of transcription J. Scholar) and human J. J. A. P. of associated proteins by electrophoresis and Scholar). proteins identified in the nuclear localization not reported These proteins are associated with the and the acid and presence of these proteins in liver nuclear extracts be to of the nuclear more to nuclear in of the of the and its to cytosolic proteins of lower of the proteins are and in a to to the in the of a nuclear localization nuclear proteins identified in the were expressed in males as with females, the differences in abundance being for all but of the proteins dimorphic hepatic expression at the mRNA protein has for of these These include the proteins A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar, S. of hepatic and regulation by growth hormone and hormone in 1991; A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. acid of acid in male and female of and 1992; 294: S. Sexual of hepatic protein mRNA expression in rats with of and P.K. Waxman D.J. Sex-dependent expression and growth hormone regulation of class alpha and class mu glutathione S-transferase mRNAs in adult rat liver..Biochem. J. 1993; 294: 159-165Google Scholar, A. C. J. of regulation of glutathione in rat liver and Sex differences and that growth hormone the hepatic J. 1992; and various A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar, of mRNA expression in male and female rats of various Biol. and the A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar, J. J. regulation of acid levels in adult rat 134: Scholar, G. J. and differences in the and of and a in rat Metab. Scholar, H. H. regulation of and by growth of growth hormone regulation in Scholar). sexually dimorphic pattern of expression of the proteins identified in and in is reported for the of the proteins identified were GH-regulated and expressed in males GH regulation at the mRNA protein has shown for of these A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar, S. of hepatic and regulation by growth hormone and hormone in 1991; A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar, of mRNA expression in male and female rats of various Biol. and A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar, H. H. regulation of and by growth of growth hormone regulation in Scholar, and of rat hepatic 1996; Scholar). GH is for the for acid K, D-like, A3, NF45, and acid GH has shown to the expression of at the of gene expression A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar, H. H. regulation of and by growth of growth hormone regulation in Scholar) and the expression of and at the gene and protein P.K. Waxman D.J. Sex-dependent expression and growth hormone regulation of class alpha and class mu glutathione S-transferase mRNAs in adult rat liver..Biochem. J. 1993; 294: 159-165Google Scholar, A. C. J. of regulation of glutathione in rat liver and Sex differences and that growth hormone the hepatic J. 1992; Scholar). In the these proteins not changes in nuclear in to GH treatment. the that GH may protein mRNA levels and/or nuclear protein abundance gel spots identified as the protein exhibited patterns of spot in and GH-treated males as with spot more in males in and not to continuous GH that is in a sex-dependent manner. In to proteins such as and which were to be sexually dimorphic and GH-regulated in the and in DNA microarray A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. of the proteins identified in the were not identified as sexually dimorphic or GH-responsive at the mRNA in DNA microarray analysis A. Clodfelter K.H. Waxman D.J. Sexual dimorphism of rat liver gene expression: Regulatory role of growth hormone revealed by deoxyribonucleic acid microarray Endocrinol. Scholar). These proteins include and for include the high of in the microarray analysis and the that GH may effects on protein localization or post-translational which not be in an analysis of mRNA of the proteins identified are Nuclear has reported for which as a DNA protein in the S. of a acid protein from rat liver as A. 1985; Scholar). displays an activity of of as an Biol. Chem. 1993; Scholar). identified D-like, and or and DNA and including mRNA and G. S. proteins and the of Rev. 1993; Scholar). NF45, a has reported to gene expression in mammalian protein nuclear factor as a and of gene expression in mammalian Biol. Scholar). is a protein of signaling that protein and activity of protein activity by localization in rat liver Scholar, S. H. of endogenous in protein regulation in the rat Scholar, S. role of endogenous in the of protein activity with of rat Scholar, of endogenous in of protein and acid in liver J. Scholar). all the proteins and the are of as are sexually GH-regulated nuclear proteins with the to gene transcription and/or and to the regulation of gene expression in liver. In acts as a that signaling and to a of growth by on and which its and and K. O. J. protein Scholar, J. K. heterogeneous nuclear protein to DNA and A. 98: Scholar, J. Growth heterogeneous nuclear expression and Biol. Chem. Scholar). of the sexually dimorphic or GH-regulated proteins identified in are transcription that the transcription factor not identified as a liver nuclear protein especially given that protein levels differ between and males Waxman D.J. growth hormone of hepatic regulation and role in liver gene a that for the individual liver nuclear extracts on gels by and not is that the of in the liver nuclear extracts low for by the SYPRO protein spots to the of were by of a gel of a male nuclear with high These spots were by SYPRO that the abundance in nuclear extracts of and that of many transcription H. S. two-dimensional and of nuclear proteins from as for of transcription J. is low for by of the nuclear with to transcription and proteins may be in In the analysis of rat liver nuclear extracts has shown that GH plays an important role in establishing sex-dependent patterns of hepatic nuclear role of GH in nuclear protein regulation is prominent the role that GH plays in the regulation of sexually dimorphic expression of rat liver is not given the large number of including protein post-translational and all of which may to sex differences in the nuclear

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