Almost all of the previous studies with growth hormone (GH) have been done with exogenously supplied GH and, therefore, involve actions of the hormone through its receptor. However, the actions of endogenous or lymphocyte GH are still unclear. In a previous study, we showed that overexpression of GH (GHo) in a lymphoid cell line resulted in protection of the cells to apoptosis mediated by nitric oxide (NO). In the present study, we show that the protection from apoptosis could be transferred to control cells with culture fluids obtained from GHo cells and blocked by antibodies to the insulin-like growth factor-1 (IGF-1) or antibodies to the IGF-1-receptor (IGF-1R). Northern and Western blot analysis detected significantly higher levels of IGF-1 in cells overexpressing GH. An increase in the expression of the IGF-1R in GHo cells was also detected by Western blot analysis, 125I-IGF-1 binding and analysis of IGF-1R promoter luciferase constructs. Transfection of GHo cells with a dominant negative IGF-1R mutant construct blocked the generation of NO and activation of Akt seen in GHo cells compared to vector alone control EL4 cells. The results suggest that one of the consequences of the overexpression of GH, in cells lacking the GH receptor, is an increase in the expression of IGF-1 and the IGF-1R which mediate the protection of EL4 lymphoma cells from apoptosis.

Ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, catalyzes the insertion of iron into protoporphyrin to form heme. This pathway provides heme for hemoglobin and other essential hemoproteins. The regulatory role of oxygen in the pathway has not been clearly established. In this study, we examined whether FECH gene expression is upregulated during hypoxia by a mechanism which involves the hypoxia-inducible factor 1 (HIF-1). Two HIF-1 binding motifs were identified within the −150 bp FECH minimal promoter sequence. Exposure of HEL, K562, and Hep-G2 cells to hypoxia for 18 hours resulted in a significant increase in FECH mRNA expression (p < 0.05). Hypoxia also transactivated the minimal promoter for the FECH gene in the cells. Transient co-expression of wild-type HIF-1α or a dominant negative HIF-1α with the FECH minimal promoter luciferase construct stimulated or blocked FECH promoter activity, respectively. Expression of the von Hippel-Lindau (VHL) tumor suppressor factor blocked the expression of both FECH mRNA and HIF-1α protein during normoxic culture of renal carcinoma cell line (RCC4). The results suggest that the FECH gene is a target for HIF-1 during hypoxia.

The antiapoptotic action of exogenous growth hormone (GH) has been reported for several lymphoid cell lines; however, the potential role of endogenous GH in apoptosis has not been thoroughly investigated. This study was designed to investigate the effects of endogenous GH on apoptosis induced by methyl methanesulfonate (MMS) in a T cell lymphoma overexpressing GH (GHo). The results of these experiments have shown that in EL4 lymphoma cells, overexpression of GH sustained viability after exposure to MMS compared to control cells. The extent of DNA fragmentation measured by ladder formation on agarose gels was reduced in GHo cells following treatment with MMS, when compared to control cells. Adding exogenous GH to control cells and treatment of GHo cells with antibodies to GH had no effect on MMS-induced DNA ladder formation. In further studies, DNA microarray analysis suggested a marked decrease in the constitutive expression of bax, BAD, and caspases 3, 8, and 9 in GHo cells compared to controls. In addition, after treatment with MMS, the activities of caspases 2, 3, 6, 8, and 9 were all lower than control in GHo cells. Western blot analysis detected an increase in Bcl-2 while the levels of nuclear factor kappa B (NFkappaB) remained unchanged in GHo cells. Treatment of EL4 cells with antisense deoxyoligonucleotides to GH and specific inhibitors of NFkappaB (SN-50) increased DNA fragmentation. GHo cells show increased levels of phosphorylated Akt and GSK-3, suggesting inactivation of this proapoptotic protein. The results, taken together with our previous data which showed increased nitric oxide formation in GHo cells, suggest a possible mechanism for the antiapoptotic effects of endogenous GH through the production of nitric oxide and support the idea that endogenous GH may play an important role in the survival of lymphocytes exposed to stressful stimuli.

Growth hormone (GH) is produced by immunocompetent cells and has been implicated in the regulation of a multiplicity of functions in the immune system involved in growth and activation. However, the actions of endogenous or lymphocyte GH and its contribution to immune reactivity when compared with those of serum or exogenous GH are still unclear. In the present study, we overexpressed lymphocyte GH in EL4 lymphoma cells, which lack the GH receptor (GHR), to determine the role of endogenous GH in nitric oxide (NO) production and response to genotoxic stress. Western blot analysis demonstrated that the levels of GH increased approximately 40% in cells overexpressing GH (GHo) when compared with cells with vector alone. The results also show a substantial increase in NO production in cells overexpressing GH that could be blocked by NG-monomethyl-l-arginine (l-NMMA), an l-arginine analogue that competitively inhibits all three isoforms of nitric oxide synthase (NOS). No evidence was obtained to support an increase in peroxynitrite in cells overexpressing GH. Overexpression of GH increased NOS activity, inducible nitric oxide synthase (iNOS) promoter activity, and iNOS protein expression, whereas endothelial nitric oxide synthase and neuronal nitric oxide synthase protein levels were essentially unchanged. In addition, cells overexpressing GH showed increased arginine transport ability and intracellular arginase activity when compared with control cells. GH overexpression appeared to protect cells from the toxic effects of the DNA alkylating agent methyl methanesulfonate. This possibility was suggested by maintenance of the mitochondrial transmembrane potential in cells overexpressing GH when compared with control cells that could be blocked by l-NMMA. Taken together, the data support the notion that lymphocyte GH, independently of the GH receptor, may play a key role in the survival of lymphocytes exposed to stressful stimuli via the production of NO.

A substantial body of research exists to support the production of growth hormone by cells of the immune system. However, the function and mechanism of action of lymphocyte-derived growth hormone remain largely unelucidated. Since, it has been found that exogenous growth hormone (GH) primes neutrophils for the production of reactive oxygen intermediates (ROI) and in particular superoxide (O-2(-)), we investigated the role of GH on the production of O-2(-) in T cells. Furthermore, we examined whether endogenous and exogenous GH act similarly. Our studies show that overexpression of GH in EL4, a T-cell lymphoma cell line, results in a decrease in the production of O-2(-) compared to control cells, as detected using the fluorescent dye, dihydroethidium. O-2(-) production in control cells was not affected by treatment with inhibitors of xanthine oxidase or a non-specific NADPH-oxidase inhibitor. However, treatment with diallyl sulfide, an inhibitor of cytochrome P450 2E1 mimicked the reduction in O-2(-) production seen in cells overexpressing GH. Although no significant change could be detected in CYP2E1 protein levels, CYP2E1 activity was found to be greater in control EL4 than in cells overexpressing GH. Both the decrease in O-2(-) production and the lower CYP2E1 activity in GH overexpressing cells could be abrogated by treatment with N-G-monomethyl-L-arginine, an inhibitor of nitric oxide synthase. The overexpression of GH protects cells from apoptosis induced by isoniazid,a CYP2E1 inducer, suggesting a role for nitric oxide as a mediator in the regulation of xenobiotic metabolism and apoptosis-protection by lymphocyte GH.

Previous work from our laboratory has shown that cells of the immune system produce a growth hormone (GH) molecule similar to that produced by the pituitary. In the present study, using Southern analysis of RT-PCR products and sequencing of cloned cDNA molecules, we demonstrate that lymphoid cell lines utilize the same promoter and first exon as the pituitary somatotrope. To identify the cis-elements involved in transcriptional regulation of immune cell-derived GH, we have coupled rat GH promoter fragments to a luciferase reporter gene and transfected a monocyte cell line (P-388) by electroporation. The results suggest the presence of both positive (–299/–193 bp) and negative (–193/–107 bp) regulatory elements. The same constructs transfected in the pituitary cell line, GH3, in contrast to the monocyte cell line, showed a gradual decrease in luciferase expression. The overexpression of GHF-1 or GHF-2 resulted in a modest but significant reduction in rat GH promoter activity in the P-388 cell line. Taken together, the data suggest that immune cells utilize the same first exon and promoter sequence for the expression of monocyte GH as that reported for the expression of pituitary GH. Further, it appears that sequences between –299 and –107 bp are important in the regulation of the promoter where different transcription factors may be recruited to promote GH expression in a monocyte cell line.

A number of studies from different laboratories clearly show that cells of the immune system produce a GH molecule indistinguishable from that produced in the pituitary. A more recent finding from our studies suggests that monocytes use the same first exon and promoter sequence for the expression of lymphocyte GH as that reported for the expression of pituitary GH. In this report we have extended these results by determining that two members of the SP family of transcription factors, SP1 and SP3, bind to the region at -138/-133 bp containing a GGGAGG motif. Confirmation that this region of the monocyte GH promoter-bound SP1 and SP3 was accomplished using electrophoretic mobility shift assays with SP1 consensus and mutant probes as well as specific antibodies to SP1 and SP3. Selective mutation of the SP1/SP3 site increased basal transcription by 73%, indicating that this site is important in transcriptional inhibition. Overexpression of SP1 had no demonstrable effect on the GH promoter, whereas overexpression of SP3 caused inhibition of expression in P-388 monocyte cells. Cotransfection of P-388 cells with overexpression vectors for both SP1 and SP3 transcription factors also resulted in inhibition of basal expression. Transfection experiments in Drosophila SL-2 cells overexpressing SP1 and/or SP3 suggest that both factors repress the basal expression of GH promoter luciferase constructs and that the effect together was additive. Taken together, the results demonstrate that basal expression of monocyte GH may be negatively regulated by SP3.

Objectives: Compare resting state regional cerebral blood flow [rCBF] in patients with fibromyalgia [FMS], non-patients with FMS, and healthy controls as a function of insidious or physically traumatic symptom onset.Methods: All subjects were women and included 21 FMS patients with insidious symptom onset; 11 FMS patients with physically traumatic onset; 13 residents recruited from the Alabama community with FMS of insidious onset who had not consulted a physician for pain in the past 10 years [non-patients]; and 29 residents recruited from the Alabama community without FMS or any other chronic pain syndromes or illnesses [healthy controls]. All subjects underwent single photon emission computed tomographic imaging of brain rCBF during resting conditions, pain threshold assessment, as well as lumbar puncture, and they completed standardized self-report measures of pain intensity, fatigue, and depression. Group differences on these assessments were evaluated by analysis of variance and the Least Significant Difference test.Results: Fibromyalgia patients with insidious pain onset showed significantly lower rCBF levels than controls in the left and right caudate nucleus [P's less than or equal to .05]. Fibromyalgia non-patients with insidious pain onset showed significantly lower rCBF than controls in the right caudate nucleus [P = .019]. Patients with either insidious or traumatic pain onset also showed significantly lower rCBF than controls in the left and right thalamus [P's less than or equal to .05]. All groups with FMS, compared to controls, were characterized by significantly lower pain threshold levels [P's less than or equal to .001] and significantly higher cerebrospinal fluid [CSF] levels of substance P [P's less than or equal to .05]. Both FMS patient groups reported significantly higher levels of fatigue and depression than the non-patients with FMS [P's less than or equal to .05] and controls [P's < .03].Conclusions: Insidious onset of FMS pain among patients and nonpatients is associated with abnormal resting state rCBF in the caudate nucleus, whereas traumatic onset of FMS pain among patients is associated with abnormal resting state rCBF in the thalamus. These differences are not associated with differences in pain threshold or CSF substance P. However, the presence of abnormal thalamic rCBF, alone or in combination with abnormal caudate rCBF is associated with greater depression and fatigue compared to abnormal caudate rCBF alone. Future studies should investigate whether abnormal thalamic and caudate responses to acute pain may also be associated with type of pain onset or with symptom severity.

Several reports support the view that growth hormone (GH) promotes proliferation and cytotoxicity by T cells in a mixed leukocyte culture (MLC). The present study was undertaken to begin to determine the mechanism of action of GH on the MLC in vitro. First, we determined that peripheral blood mononuclear cells (PBMC) cultured with mitomycin-treated allogeneic PBMC in an MLC in the presence of exogenously added rhGH develop an augmented proliferative (25-100%) and cytotoxic response (50-600%). We next examined the possibility that GH may promote alloresponses by inducing gamma-interferon (IGN gamma) production. In these experiments, in situ hybridization was used to determine the frequency of cells expressing mRNA for IFN gamma. It was observed that GH increased significantly the frequency of cells expressing mRNA for IFN gamma (100-800%). To determine the site of action of rhGH, we evaluated the response of purified T cells to alloantigens in the presence of rhGH. The addition of rhGH to an MLC had no demonstrable effect when purified T cells were used as the responding population. However, when T cells were reconstituted with autologous mitomycin-treated PBMC and used as the responding population, rhGH augmented proliferation and cytotoxicity. Taken together, these data show that rhGH augments proliferation, cytotoxicity and IFN gamma production during an MLC, and at lease part of the action of rhGH appears to be on the autologous antigen-presenting cell.

Reciprocal communication between the neuroendocrine and immune systems is critical to the establishment of host homeostatic and defence mechanisms. The production and utilisation of common ligands and their receptors by cells of the immune and neuroendocrine systems constitutes a biochemical information circuit between and within the immune and neuroendocrine systems. Although the structures of the various signalling components appear to be similar in both systems, the regulation of their synthesis may be different. Growth hormone and prolactin have similar and marked influences on the function/activity of each of the major immune cell types, both in vitro and in vivo. The underlying molecular mechanisms are just beginning to be unravelled, and it is anticipated that further work in this rapidly developing field will establish abnormal pituitary and/or lymphocyte growth hormone and prolactin synthesis and function as a contributory factor to a number of pathologic situations, including leukaemia and autoimmunity.

The messenger RNA for rat growth hormone (GH) was isolated from rat spleen lymphocytes and cloned as a cDNA after PCR. Four clones, including part of the prehormone sequence and the full-length mature GH sequence, were obtained and sequenced. The sequence data revealed that rat lymphocyte GH was identical to that reported for pituitary GH. The results do not support the expression by alternate splicing of the 20-kD variant described in the pituitary. We also cloned the promoter of lymphocyte GH and analyzed its nucleotide sequence including 300 base pairs of the 5'-flanking region. The promoter sequence we obtained did not exactly match that reported in the literature because of reading compressions on the gel. However, parellel sequencing of thymus, spleen and pituitary GH promoter sequences gave identical patterns of compressions in the gel. The results suggest that the sequence in all the tissues was the same. Collectively, these studies demonstrate that lymphocytes express authentic GH mRNA and suggest that GH gene expression may be regulated in immune tissue by similar transcriptional factors to those described in the pituitary.