Responsiveness of an SV40-immortalized hepatocyte cell line to growth hormone

Kempe, Kirsten; Isom, Harriet C.; Greene, Frank E.

doi:10.1016/0006-2952(95)98506-5

Cited by 22 publications

(22 citation statements)

References 37 publications

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“…Rabbit anti-GHR C-terminal domain polyclonal antibody (pAb) 2941 (45) Cell Culture and Transfection-CWSV-1, a GH-responsive SV40-immortalized cell line derived from adult male rat hepatocytes (47), was maintained and passaged in RPCD medium with the addition of a trace element mixture ("complete RPCD medium") (40). Transfections were carried out as described previously (30) using FuGENE 6 transfection reagent (Roche Applied Science, Mannheim, Germany) at 2 l of FuGENE 6 g of total DNA.…”

Section: Methodsmentioning

confidence: 99%

Role of the Cytokine-induced SH2 Domain-containing Protein CIS in Growth Hormone Receptor Internalization

Landsman

Waxman

2005

Journal of Biological Chemistry

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The cytokine-inducible SH2 domain-containing protein CIS inhibits signaling from the growth hormone (GH) receptor (GHR) to STAT5b by a proteasome-dependent mechanism. Here, we used the GH-responsive rat liver cell line CWSV-1 to investigate the role of CIS and the proteasome in GH-induced GHR internalization. Cell-surface GHR localization and internalization were monitored in GH-stimulated cells by confocal immunofluorescence microscopy using an antibody directed against the GHR extracellular domain. In GH naïve cells, GHR was detected in small, randomly distributed granules on the cell surface and in the cytoplasm, with accumulation in the perinuclear area. GH treatment induced a rapid (within 5 min) internalization of GH⅐GHR complexes, which coincided with the onset of GHR tyrosine phosphorylation and the appearance in the cytosol of distinct granular structures containing internalized GH. GHR signaling to STAT5b continued for ϳ30 -40 min, however, indicating that GHR signaling and deactivation of the GH⅐GHR complex both proceed from an intracellular compartment. The internalization of GH and GHR was inhibited by CIS-R107K, a dominant-negative SH2 domain mutant of CIS, and by the proteasome inhibitors MG132 and epoxomicin, which prolong GHR signaling to STAT5b. GH pulse-chase studies established that the internalized GH⅐GHR complexes did not recycle back to the cell surface in significant amounts under these conditions. Given the established specificity of CIS-R107K for blocking the GHR signaling inhibitory actions of CIS, but not those of other SOCS/CIS family members, these findings implicate CIS and the proteasome in the control of GHR internalization following receptor activation and suggest that CIS-dependent receptor internalization is a prerequisite for efficient termination of GHR signaling. Growth hormone (GH)2 is an important regulator of somatic growth and cellular metabolism. GH exerts its action via the GH receptor (GHR), a transmembrane protein of the cytokine receptor superfamily that activates multiple intracellular signaling pathways, including the STAT, mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C pathways (1, 2). Upon binding of ligand, the dimeric GHR undergoes a conformational change (3, 4) and induces tyrosine phosphorylation, resulting in activation of the tyrosine kinase JAK2, which catalyzes tyrosine phosphorylation of GHR and of GHR-or JAK2-associated STAT1, STAT3, STAT5a, and STAT5b (5, 6). The tyrosine-phosphorylated STAT proteins dimerize and translocate to the nucleus, where they bind to specific DNA response elements upstream of GH target genes and activate gene transcription (7,8).Many physiological responses to GH are dependent on the temporal pattern of plasma GH stimulation, which is sex-dependent and subject to neuroendocrine control. In the rat model, the adult male plasma GH pattern is characterized by regular pulses of hormone every ϳ3.5-4 h, separated by well defined GH-free intervals, whereas a more continuous plasma GH profile i...

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Section: Methodsmentioning

confidence: 99%

Role of the Cytokine-induced SH2 Domain-containing Protein CIS in Growth Hormone Receptor Internalization

Landsman

Waxman

2005

Journal of Biological Chemistry

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“…The CWSV-1 hepatocytes were chosen because the cells express GHR, synthesize IGF-I in response to GH (32,37,54), and transduce GH signals via the JAK/STAT pathway (22,32,45). Incubation of hepatocytes with TNF-␣ for 4 h completely inhibited the synthesis of IGF-I mRNA induced by GH but had no effects on IGF-I mRNA content under basal, nonstimulated conditions.…”

Section: Discussionmentioning

confidence: 99%

“…of Microbiology, College of Medicine, Pennsylvania State University). CWSV-1 is an SV40 transformed rat hepatocyte cell line that has been extensively characterized and demonstrates many of the important regulatory mechanisms observed in normal liver tissue (32,37,55). CWSV-1 cells were grown in RPCD medium (32,36,53) for 48 h. TNF-treated cells were incubated with 10 ng/ml TNF-␣ for 4 h, and then 500 ng/ml rhGH were added for the indicated time periods.…”

Section: Methodsmentioning

confidence: 99%

“…To further investigate the complex events that occur in vivo during the septic insult and to dissect the role of individual cytokines, we examined the effects of TNF-␣ on IGF-I synthesis by CWSV-1 hepatocytes. Of importance, CWSV-1 hepatocytes synthesize IGF-I mRNA in response to GH, and GH signaling in this cell line has been extensively characterized (23,32,45). The time course of IGF-I mRNA expression after GH administration was determined using 500 ng/ml GH (Fig.…”

Section: Role Of Tnf In Mediating Sepsis-induced Changes In Gh/igf-i mentioning

confidence: 99%

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Tumor necrosis factor mediates hepatic growth hormone resistance during sepsis

Yumet¹,

Shumate²,

Bryant³

et al. 2002

American Journal of Physiology-Endocrinology and Metabolism

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During sepsis, growth hormone (GH) resistance contributes to the catabolism of muscle protein. To determine the role of tumor necrosis factor (TNF) as a mediator of GH resistance, we examined the effects of a TNF antagonist [TNF-binding protein (TNFbp)] on the GH/insulin-like growth factor (IGF) I system during abdominal sepsis. To investigate potential mechanisms, the effects of TNF on the IGF-I response to GH and GH signaling were examined in cultured rat hepatocytes (CWSV-1). Three groups of rats were studied: Control, Sepsis, and Sepsis + TNFbp. Liver, gastrocnemius, and plasma were collected on day 5. In gastrocnemius, neither sepsis nor TNFbp altered the abundance of IGF-I mRNA. However, septic rats demonstrated an increase in circulating GH and a reduction in plasma IGF-I concentrations that was ameliorated by pretreatment with TNFbp. Liver from septic rats demonstrated a 50% reduction in GH receptor (GHR) and IGF-I mRNA on day 5 that was attenuated by TNFbp. However, the abundance of GHR protein was not different in liver from Control, Sepsis, or Sepsis + TNFbp rats. Consequently, a decreased amount of hepatic GHR does not explain the GH-resistant septic state. In CWSV-1 hepatocytes, TNF-α had no effect on GHR protein level but inhibited the induction of IGF-I mRNA by GH. Nuclear protein from TNF-treated hepatocytes demonstrated similar levels of phosphorylated signal transducer and activator of transcription-5 (STAT5) and DNA binding relative to controls 5 min after GH treatment. However, both of these parameters were decreased (vs. control) in TNF-treated cells 60 min after GH treatment. Collectively, these results suggest that TNF mediates hepatic GH resistance during sepsis by inhibiting the duration of signaling via the janus kinase-2/STAT5 pathway.

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“…model (21), have revealed that phosphotyrosine phosphatases (PTPs) play an important role in GH signal termination (22). Rapid deactivation of GH-activated STAT5b following cessation of a GH pulse was shown to involve STAT5b phosphotyrosine dephosphorylation as an essential first step.…”

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confidence: 99%

Interaction of Growth Hormone-activated STATs with SH2-containing Phosphotyrosine Phosphatase SHP-1 and Nuclear JAK2 Tyrosine Kinase

Ram

Waxman

1997

Journal of Biological Chemistry

190

143

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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 GHactivated 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.

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Responsiveness of an SV40-immortalized hepatocyte cell line to growth hormone

Cited by 22 publications

References 37 publications

Role of the Cytokine-induced SH2 Domain-containing Protein CIS in Growth Hormone Receptor Internalization

Role of the Cytokine-induced SH2 Domain-containing Protein CIS in Growth Hormone Receptor Internalization

Tumor necrosis factor mediates hepatic growth hormone resistance during sepsis

Interaction of Growth Hormone-activated STATs with SH2-containing Phosphotyrosine Phosphatase SHP-1 and Nuclear JAK2 Tyrosine Kinase

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