06-554 Sigma-AldrichAnti-STAT5B Antibody

We previously reported the presence of functional human GH receptors (hGHRs) in the human fetal hepatocyte (FH) as early as the first trimester. Interestingly, fetal serum levels of hGH are in the acromegalic range, yet certain hGH-dependent factors are expressed at very low levels (IGF-I, IGF-binding protein-3), suggesting that fetal liver has limited responsiveness to hGH. To determine whether this is due to the fetal tissue levels of hGHR or factors in the hGH/hGHR axis that might influence hGHR function, we compared hGHR isoforms and downstream signaling proteins in FH versus human adult liver (HAL). Immunoprecipitation/immunoblotting (IB) analyses found similar precursor and mature hGHR forms while RT-PCR assays of truncated (T) hGHR(1-279), dominant negative for the full-length (FL) receptor, showed similar T/FL mRNA ratios in FH and HAL. IB demonstrated that Janus kinase (JAK) 2, signal transducers and activators of transcription (STAT(1, 3, 5A/B)), and suppressors of cytokine signaling (SOCS(1, 2, 3, cytokine-inducible SH2-containing protein (CIS))) proteins were detectable in all FH and HAL tested (12 weeks of fetal age to 60 years); the levels were similar (STAT5B) or lower (JAK2/STAT1/STAT3/STAT5A: 38-53%, SOCS/CIS: 58-76%) in FH compared with HAL. Our studies to date demonstrate that, during hepatocyte development, hGHR levels are lower in the fetal cells but the hGHR isoforms, including the relative amount of truncated versus FL, remain unchanged. The JAK2/STAT/SOCS signaling molecules are present in the FH as early as the first trimester. However, they are generally at less than 50% level in postnatal liver. These data suggest that low expression of both hGHR and major hGHR signaling components may explain the limited responsiveness of the fetal cells to the high circulating levels of hGH.

The consequences of T-cell activation depend exclusively on costimulation during antigen-T-cell receptor interaction. Interaction between the T-cell coreceptor CD28 and its ligand B7 during antigen-antigen receptor engagement results in full activation of T cells, the outcomes of which are proliferation and effector functions. The ability of CD28 to costimulate the production of interleukin-2 (IL-2) explains the importance of this costimulation. The signaling event mediated by CD28 engagement has been proposed to have 2 components: one is sensitive to the immunosuppressive drug cyclosporin A (CsA), and the other one is CsA-resistant. In this report, we demonstrate that the CsA-resistant pathway is sensitive to the immunosuppressive drug rapamycin. Treatment with rapamycin blocked IL-2 production after activation of human peripheral blood T cells with phorbol ester (PMA) and anti-CD28 (CsA-resistant pathway), whereas this drug did not have any effect on PMA plus ionomycin stimulation (CsA-sensitive pathway). The inhibitory effect of rapamycin was on messenger RNA stability and translation, rather than on IL-2 transcription or protein turnover.

The transcription factors signal transducer and activator of transcription (Stat)5a and Stat5b have been implicated in the GH regulation of CYP2C genes in rodent liver. In addition to full-length Stat5 isoforms, truncated Stat5 proteins (Stat5beta), lacking the transactivating domain, have been demonstrated. In this study we found that Stat5beta can be formed by proteolytic cleavage in rat liver nuclei and that the activity of the protease is independent of GH. The GH regulation of the female-specific CYP2C12 gene has recently been shown to be conveyed by two adjacent Stat5-binding elements in the 5'-upstream region. We found that binding of Stat5 in liver nuclear extracts to this site involved simultaneous binding of two Stat5 dimers, most likely both Stat5b homodimers and Stat5a/Stat5b heterodimers. We also investigated Stat5 binding to a potential composite Stat5 element in the 3'-untranslated region (UTR) of CYP2C12. Several Stat5 complexes were formed on this element including Stat5beta-containing complexes. In transient transfection experiments we could demonstrate that the 3'-UTR element reduced GH activation of a CYP2C12-luciferase reporter construct harboring the 5'-Stat5 elements. We speculate that binding of Stat5beta to the 3'-UTR element could be of relevance for the GH-dependent and sex-specific expression of CYP2C12.

We have isolated a second human Stat5 cDNA, Stat5B, and demonstrated that the genes encoding both Stat5A and Stat5B are located at chromosome 17q11.2. Both genes were constitutively transcribed in peripheral blood lymphocytes. By using specific antisera, we demonstrated that both Stat5A and Stat5B are activated by interleukin-2 (IL-2) in peripheral blood lymphocytes, natural killer-like YT leukemia cells, and human T cell lymphotropic virus type I-transformed MT-2 T cells. In COS-7 cells, which constitutively express the Janus family tyrosine kinase Jak1, reconstitution of IL-2-induced Stat5A and Stat5B DNA binding activities was dependent on the coexpression of Jak3 along with the IL-2 receptor beta chain and the common cytokine receptor gamma-chain. This IL-2-induced Stat5 activation was dependent on the presence of either of two tyrosines (Tyr-392 or Tyr-510) in the IL-2 receptor beta chain, indicating that either of these two tyrosines can serve as a docking site. Moreover, we demonstrated that human Stat5 activation is also dependent on Tyr-694 in Stat5A and Tyr-699 in Stat5B, indicating that these tyrosines are required for dimerization. The COS-7 reconstitution system described herein provides a valuable assay for further elucidation of the IL-2-activated JAK-STAT pathway.