Jessica Schwartz

GROWTH HORMONE STIMULATES PHOSPHORYLATION AND ACTIVATION OF ELK-1 AND EXPRESSION OF C-FOS, EGR-1, AND JUNB THROUGH ACTIVATION OF EXTRACELLULAR SIGNAL- REGULATED KINASES 1 AND 2

Growth hormone (GH), a major regulator of normal body growth and metabolism, regulates cellular gene expression. The transcription factors Elk-1 and Serum Response Factor are necessary for GH-stimulated transcription of c-fos through the Serum Response Element (SRE). GH stimulates the serine phosphorylation of Elk-1, thereby enabling Elk-1 to mediate transcriptional activation. The contribution of the Ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway to Elk-1-mediated transcriptional activation of the c-fos SRE in response to GH was examined. The MEK inhibitor PD098059 attenuated GH-induced expression of the endogenous SRE-regulated genes c-fos, egr-1, and junB as well as transcriptional activation mediated by the c-fos promoter. The MEK inhibitor blocked GH-stimulated activation of MEK, phosphorylation of ERK1/ERK2, and MAP kinase activity in 3T3-F442A cells. Blocking MEK activation prevented GH-induced phosphorylation of Elk-1, as well as the ability of Elk-1 to mediate transcriptional activation in response to GH. Overexpression of dominant-negative Ras or the ERK-specific phosphatase, mitogen-activated protein kinase phosphatase-1, blocked the Ras/MEK/ERK pathway and abrogated GH-induced phosphorylation of Elk-1. GH failed to stimulate phosphorylation or activation of Jun N-terminal kinase under the conditions used. GH slightly increased p38-mediated mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2 activity, but the p38 inhibitor SB203580 did not attenuate GH-promoted Elk-1 phosphorylation. Wortmannin, which inhibited GH-induced ERK phosphorylation, also attenuated transcriptional activation of c-fos by GH. Taken together, these data suggest that GH-dependent activation of the Ras/MEK/ERK pathway and subsequent serine phosphorylation of Elk-1 contribute to GH-stimulated c-fos expression through the SRE.

Growth hormone alters lipolysis and hormone-sensitive lipase activity in 3T3-F442A adipocytes

While growth hormone (GH) has long been known as a lipolytic hormone, it has been difficult to study the cellular mechanisms for this effect. Since cultured 3T3-F442A adipocytes have recently proven to be useful to study chronic effects of GH on adipocyte metabolism, we examined the effects of GH on lipolysis. In these cells, GH alone produced a dose-dependent increase in the release of glycerol after 24 to 48 hours. The maximum increase occurred with 10 ng/mL human GH. The effect of GH was similar in the presence and absence of dexamethasone. Under each condition, the stimulation of glycerol release was accompanied by a GH-induced increase in the activity of hormone-sensitive lipase (HSL), a key lipolytic enzyme. The increase in HSL required 24 hours with GH and lasted at least 48 hours. The increase in HSL activity by epinephrine, like glycerol release, was potentiated by GH. Although GH potently simulates the activity of the lipogenic enzyme glycerol phosphate dehydrogenase (GPD) in differentiating 3T3-F442A preadipocytes, GH had a negligible effect on GPD activity in the differentiated adipocytes with chronic or short-term incubation. However, in contrast to the chronic effect of GH, short-term (30-minute) incubation with GH inhibited epinephrine-stimulated glycerol release, a characteristic transient antilipolytic effect of GH. These studies indicate that chronic GH treatment is lipolytic in cultured 3T3-F442A adipocytes, and document that lipolytic responses to GH involve an increase in the activity of HSL.

CCAAT/Enhancer-binding Protein (C/EBP) β Is Acetylated at Multiple Lysines ACETYLATION OF C/EBPβ AT LYSINE 39 MODULATES ITS ABILITY TO ACTIVATE TRANSCRIPTION

Transcription factor function can be modulated by post-translational modifications. Because the transcription factor CCAAT/enhancer-binding protein (C/EBP) β associates with the nuclear coactivator p300, which contains acetyltransferase activity, acetylation of C/EBPβ was examined to understand its regulation and function. C/EBPβ is acetylated by acetyltransferases p300 and p300/CREB-binding protein associated factor. Endogenous C/EBPβ in 3T3-F442A preadipocytes is also recognized by an acetyl-lysine-specific antibody. Analysis of truncations of C/EBPβ and peptides based on C/EBPβ sequences identified multiple lysines within C/EBPβ that can be acetylated. Among these, a novel acetylation site at lysine 39 of C/EBPβ was identified. Mutation of Lys-39 to arginine or alanine impairs its acetylation and the ability of C/EBPβ to activate transcription at the promoters for C/EBPα and c-fos. Different C/EBPβ-responsive promoters require different patterns of acetylated lysines in C/EBPβ for transcription activation. Furthermore, C/EBPβ acetylation was increased by growth hormone, and mutation of Lys-39 impaired growth hormone-stimulated c-fos promoter activation. These data suggest that acetylation of Lys-39 of C/EBPβ, alone or in combination with acetylation at other lysines, may play a role in C/EBPβ-mediated transcriptional activation.

Growth hormone regulates ternary complex factors and serum response factor associated with the c-fos serum response element

For insight into the mechanisms of gene regulation by growth hormone (GH), the regulation of transcription factors associated with the serum response element (SRE) located upstream of c-fos was examined. The SRE can mediate induction of reporter expression in response to GH. For insight into the mechanism by which GH regulates transcription factors, regulation of SRE-associated proteins by GH was examined. In nuclear extracts from 3T3-F442A fibroblasts, several SRE-binding complexes were identified by electrophoretic mobility shift assay. GH treatment for 2–10 min transiently increased binding of two complexes; binding returned to control values within 30 min. The two GH-stimulated complexes were supershifted by antibodies against the serum response factor (SRF), indicating that they contained SRF or an antigenically related protein. One of the GH-stimulated complexes was supershifted by antibody against Elk-1, suggesting that it contains a ternary complex factor (TCF) such as Elk-1 in addition to SRF. Induction of binding by GH was lost when the SRF binding site in the SRE was mutated, and mutation of either the SRF or TCF binding site altered the pattern of protein binding to the SRE. Mutation of the SRF or TCF binding site in SRE-luciferase plasmids inhibited the ability of GH to stimulate reporter expression, supporting a role for both SRF and TCF in GH-induced transcription of c-fos via the SRE. The TCF family member Elk-1 is capable of mediating GH-stimulated transcription, since GH-stimulated reporter expression was mediated by the transcriptional activation domain of Elk-1. Consistent with this stimulation, GH rapidly and transiently stimulated the serine phosphorylation of Elk-1. The increase was evident within 10 min and subsided after 30 min. Taken together, these data indicate that SRF and TCF contribute to GH-promoted transcription of c-fos via the SRE and are consistent with GH-promoted phosphorylation of Elk-1 contributing to GH-promoted transcriptional activation via the SRE.

Role of Wnt10b and C/EBPα in spontaneous adipogenesis of 243 cells☆☆☆

Abstract This report examines the balance of positive and negative adipogenic factors in a line of immortalized 243 embryonic fibroblasts that undergo spontaneous preadipocyte differentiation. Control of adipogenesis reflects the interplay of factors that promote or inhibit expression of C/EBPα and PPARγ. The 243 cells express C/EBPα early and at elevated levels compared to 3T3-F442A preadipocytes or adipocytes. Cell clones were derived from the heterogeneous 243 population for ability or inability to differentiate into adipocytes. Wnt10b, a secreted protein that inhibits adipogenesis, is expressed at high levels in cells with low adipogenic potential and is undetectable in preadipocytes that spontaneously differentiate. In contrast, C/EBPα is expressed at reduced levels in cells with low adipogenic potential, and is expressed at high levels in preadipocytes that spontaneously differentiate. These data are consistent with a model in which decreased Wnt10b, coupled with increased C/EBPα, results in induction of PPARγ and spontaneous adipogenesis of 243 cells.

CCAAT/Enhancer-binding Protein β (C/EBPβ) and C/EBPδ Contribute to Growth Hormone-regulated Transcription of c-fos

Using the c-fos enhancer as a model to analyze growth hormone (GH)-promoted gene expression, the roles of CCAAT/enhancer-binding proteins (C/EBPs) in GH-regulated transcription were investigated. In 3T3-F442A fibroblasts stably expressing the c-fos promoter mutated at the C/EBP binding site upstream of luciferase, c-fos promoter activity is stimulated by GH 6–7-fold; wild type c-fos promoter shows only a 2-fold induction by GH. This suggests that C/EBP restrains GH-stimulated expression of c-fos. Electrophoretic mobility shift assays with nuclear extracts from 3T3-F442A cells indicate that GH rapidly (2–5 min) increases binding of C/EBPβ and C/EBPδ, to the c-fos C/EBP binding site. Both liver activating protein (LAP) and liver inhibitory protein (LIP), forms of C/EBPβ, are detected in 3T3-F442A cells by immunoblotting. GH increases the binding of LAP/LAP and LAP/LIP dimers. Overexpression of LIP interferes with GH-promoted reporter expression in CHO cells expressing GH receptors, consistent with the possibility that LIP restrains GH-stimulated c-fos expression. Overexpression of LAP elevates basal luciferase activity but does not influence promoter activation by GH, while overexpressed C/EBPδ elevates basal promoter activity and enhances the stimulation by GH. GH stimulates the expression of mRNA for C/EBPβ and -δ and increases levels of C/EBPδ. Although C/EBPβ is not detectably altered, GH induces a shift to more rapidly migrating forms of LIP and LAP upon immunoblotting. Treatment of extracts from GH-treated cells with alkaline phosphatase causes a shift of the slower migrating form to the rapidly migrating form, consistent with GH promoting dephosphorylation of LIP and LAP. These studies implicate C/EBPβ and -δ in GH-regulated gene expression. They also indicate that GH stimulates the binding of C/EBPβ and -δ to the c-fos promoter and promotes the dephosphorylation of LIP and LAP. These events may contribute to the ability of C/EBPβ and -δ to regulate GH-stimulated expression of c-fos.

Profiles of Growth Hormone (GH)-regulated Genes Reveal Time-dependent Responses and Identify a Mechanism for Regulation of Activating Transcription Factor 3 By GH

In examination of mechanisms regulating metabolic responses to growth hormone (GH), microarray analysis identified 561 probe sets showing time-dependent patterns of expression in GH-treated 3T3-F442A adipocytes. Biological functions significantly over-represented among GH-regulated genes include regulators of transcription at early times, and lipid biosynthesis, cholesterol biosynthesis, and mediators of immune responses at later times (48 h). One novel GH-induced gene encodes activating transcription factor 3 (ATF3). Atf3 mRNA expression and promoter activity were stimulated by GH. Genes for ATF3 and growth arrest and DNA damage-inducible gene 45 gamma (GADD45γ) showed similar time-dependent patterns of responses to GH, suggesting similar regulatory mechanisms. A conserved sequence in the promoters of the Atf3 and Gadd45γ genes contains a CCAAT/enhancer-binding protein (C/EBP) site previously observed in the Gadd45γ promoter, suggesting a novel corresponding C/EBP site in the Atf3 promoter. C/EBPβ was found to bind to the predicted Atf3 C/EBP site, and C/EBPβ enhanced the activation of the wild-type Atf3 promoter. Mutation of the predicted Atf3 C/EBP site disrupted Atf3 promoter activation not only by C/EBPβ but also by GH. These findings suggest that GH regulates transcription of Atf3 through a mechanism utilizing factors, such as C/EBPβ, which bind to a novel C/EBP site.

Acetylation and deacetylation regulate CCAAT/enhancer binding protein β at K39 in mediating gene transcription

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) contains multiple acetylation sites, including lysine (K) 39. Mutation of C/EBPbeta at K39, an acetylation site in the transcriptional activation domain, impairs transcription of C/EBPbeta target genes in a dominant-negative fashion. Further, K39 of C/EBPbeta can be deacetylated by HDAC1, and HDAC1 may decrease C/EBPbeta-mediated transcription, suggesting that acetylation of C/EBPbeta at K39 is dynamically regulated in mediating gene transcription. Acetylation of endogenous C/EBPbeta at K39 is detected in adipose tissue, and also occurs in 3T3-L1 cells undergoing adipocyte conversion. In addition, mutation of K39 in C/EBPbeta impairs activation of its target genes encoding C/EBPalpha and PPARgamma, essential mediators of adipogenesis, as well as adipocyte genes for leptin and Glut4. These findings suggest that acetylation of C/EBPbeta at K39 is an important and dynamic regulatory event that contributes to its ability to transactivate target genes, including those associated with adipogenesis and adipocyte function.

Regulation of Glucose Transport and c-fos and egr-1 Expression in Cells with Mutated or Endogenous Growth Hormone Receptors

To identify mechanisms by which GH receptors (GHR) mediate downstream events representative of growth and metabolic responses to GH, stimulation by GH of c-fos and egr-1 expression and glucose transport activity were examined in Chinese hamster ovary (CHO) cells expressing mutated GHR. In CHO cells expressing wild-type GHR (GHR1–638), GH stimulated the expression of c-fos and egr-1, and stimulated 2-deoxyglucose uptake, responses also mediated by endogenous GHR in 3T3-F442A cells. Deletion of the proline-rich box 1 of GHR (GHRΔP) abrogated all of these responses to GH, indicating that box 1, a site of association of GHR with the tyrosine kinase JAK2, is crucial for these GH-stimulated responses. As the C-terminal half of the cytoplasmic domain of GHR is required for GH-stimulated calcium flux and for stimulation of spi-2.1 transcription, GHR lacking this sequence (GHR1–454) were examined. Not only did GHR1–454 mediate stimulation of c-fos and egr-1 expression and 2-deoxyglucose uptake, but they also media...

Growth hormone signaling pathways.

Over 20years ago, our laboratory showed that growth hormone (GH) signals through the GH receptor-associated tyrosine kinase JAK2. We showed that GH binding to its membrane-bound receptor enhances binding of JAK2 to the GHR, activates JAK2, and stimulates tyrosyl phosphorylation of both JAK2 and GHR. The activated JAK2/GHR complex recruits a variety of signaling proteins, thereby initiating multiple signaling pathways and cellular responses. These proteins and pathways include: 1) Stat transcription factors implicated in the expression of multiple genes, including the gene encoding insulin-like growth factor 1; 2) Shc adapter proteins that lead to activation of the grb2-SOS-Ras-Raf-MEK-ERK1,2 pathway; 3) insulin receptor substrate proteins implicated in the phosphatidylinositol-3-kinase and Akt pathway; 4) signal regulatory protein α, a transmembrane scaffold protein that recruits proteins including the tyrosine phosphatase SHP2; and 5) SH2B1, a scaffold protein that can activate JAK2 and enhance GH regulation of the actin cytoskeleton. Our recent work has focused on the function of SH2B1. We have shown that SH2B1β is recruited to and phosphorylated by JAK2 in response to GH. SH2B1 localizes to the plasma membrane, cytoplasm and focal adhesions; it also cycles through the nucleus. SH2B1 regulates the actin cytoskeleton and promotes GH-dependent motility of RAW264.7 macrophages. Mutations in SH2B1 have been found in humans exhibiting severe early-onset childhood obesity and insulin resistance. These mutations impair SH2B1 enhancement of GH-induced macrophage motility. As SH2B1 is expressed ubiquitously and is also recruited to a variety of receptor tyrosine kinases, our results raise the possibility that effects of SH2B1 on the actin cytoskeleton in various cell types, including neurons, may play a role in regulating body weight.