Identification of a novel cell type-specific intronic enhancer of macrophage migration inhibitory factor (MIF) and its regulation by mithramycin.

The aim of this study was to determine the genetic regulation of macrophage migration inhibitory factor (MIF). DNase I hypersensitivity was used to identify potential hypersensitive sites (HS) across the MIF gene locus. Reporter gene assays were performed in different human cell lines with constructs containing the native or mutated HS element. Following phylogenetic and transcription factor binding profiling, electrophoretic mobility shift assay (EMSA) and RNA interference were performed and the effects of incubation with mithramycin, an antibiotic that binds GC boxes, were also studied. An HS centred on the first intron of MIF was identified. The HS acted as an enhancer in human T lymphoblasts (CEMC7A), human embryonic kidney cells (HEK293T) and human monocytic cells (THP-1), but not in a fibroblast-like synoviocyte (FLS) cell line (SW982) or cultured FLS derived from rheumatoid arthritis (RA) patients. Two cis-elements within the first intron were found to be responsible for the enhancer activity. Mutation of the consensus Sp1 GC box on each cis-element abrogated enhancer activity and EMSA indicated Sp1 binding to one of the cis-elements contained in the intron. SiRNA knock-down of Sp1 alone or Sp1 and Sp3 together was incomplete and did not alter the enhancer activity. Mithramycin inhibited expression of MIF in CEMC7A cells. This effect was specific to the intronic enhancer and was not seen on the MIF promoter. These results identify a novel, cell type-specific enhancer of MIF. The enhancer appears to be driven by Sp1 or related Sp family members and is highly sensitive to inhibition via mithramycin.

Familial glucocorticoid resistance caused by a novel frameshift glucocorticoid receptor mutation.

CONTEXT: Familial glucocorticoid resistance is a rare condition with a typical presentation of women with hirsutism and hypertension, with or without hypokalemia. OBJECTIVE: The aim was to determine the cause of apparent glucocorticoid resistance in a young woman. PATIENTS AND METHODS: We studied a family with a novel glucocorticoid receptor (GR) mutation and a surprisingly mild phenotype. Their discovery resulted from serendipitous measurement of serum cortisol with little biochemical or clinical evidence for either hyperandrogenism or mineralocorticoid excess. RESULTS: The causative mutation was identified as a frameshift mutation in exon 6. Transformed peripheral blood lymphocytes were generated to analyze GR expression in vitro. Carriers of the mutation had less full-length GR, but the predicted mutant GR protein was not detected. However, this does not exclude expression in vivo, and so the mutant GR (Δ612GR) was expressed in vitro. Simple reporter gene assays suggested that Δ612GR has dominant negative activity. Δ612GR was not subject to ligand-dependent Ser211 phosphorylation or to ligand-dependent degradation. A fluorophore-tagged construct showed that Δ612GR did not translocate to the nucleus in response to ligand and retarded translocation of the wild-type GR. These data suggest that Δ612GR is not capable of binding ligand and exerts dominant negative activity through heterodimerization with wild-type GR. CONCLUSION: Therefore, we describe a novel, naturally occurring GR mutation that results in familial glucocorticoid resistance. The mutant GR protein, if expressed in vivo, is predicted to exert dominant negative activity by impairing wild-type GR nuclear translocation.

Functional evaluation of TNFAIP3 (A20) in rheumatoid arthritis.

OBJECTIVES: To determine the protein expression of TNFAIP3 in synovium and to show the capability of 6q23 intergenic SNPs, associated with rheumatoid arthritis (RA) susceptibility, to influence TNFAIP3 gene transcription. METHODS: Immunohistochemistry for TNFAIP3, NF-kB p65 and phosphorylated NF-kB p65 protein expression was performed in 6 RA knee joint synovium samples compared to 9 osteoarthritis (OA) samples. Luciferase reporter gene assays were used to examine the regulatory ability of RA associated SNP variants on TNFAIP3 promoter activity. Sense and antisense constructs were prepared for rs6920220 alleles, together with each of the 4 SNPs in r2=1 with it (rs6933404, rs2327832, rs6927172 and rs17264332), coupled to the TNFAIP3 promoter. Transient transfections were performed in a human T lymphoblastoid (CEMC7A) cell line. Bioinformatic software was utilised to prioritise SNPs for further investigation. Electrophoretic mobility shift assays (EMSA), using CEMC7A nuclear extracts, were conducted for the rs6927172 SNP alleles. RESULTS: TNFAIP3 protein expression was seen in the synovium samples and differential TNFAIP3 protein expression between RA vs. OA synoviocytes observed. Within RA synoviocytes TNFAIP3 expression is predominately cytoplasmic, whereas in OA its expression is strongly nuclear and cytoplasmic. For 3 of the 5 SNPs investigated (rs6920220, rs6933404, rs6927172) evidence of repressor activity of TNFAIP3 transcription was seen and EMSA data showed evidence of differential transcription factor binding to rs6927172 alleles. CONCLUSIONS: This is the first observation of TNFAIP3 protein expression in RA and OA synovium. In vitro analysis of 6q23 intergenic SNPs supports the possibility of the functional regulation of TNFAIP3.

Interferon-inducible factor 16 is a novel modulator of glucocorticoid action.

Previously, we used cDNA expression profiling to identify genes associated with glucocorticoid (Gc) sensitivity. We now identify which of these directly influence Gc action. Interferon-inducible protein 16 (IFI16), bone morphogenetic protein receptor type II (BMPRII), and regulator of G-protein signaling 14 (RGS14) increased Gc transactivation, whereas sialyltransferase 4B (SIAT4B) had a negative effect. Amyloid beta (A4) precursor-protein binding, family B, member 1 (APBB1/Fe65) and neural cell expressed developmentally down-regulated 9 (NEDD9) were without effect. Only IFI16 potentiated Gc repression of NF-kappaB. In addition, IFI16 affected basal expression, and Gc induction of endogenous target genes. IFI16 did not affect glucocorticoid receptor (GR) expression, ligand-dependent repression of GR expression, or the ligand-dependent induction of GR phosphorylation on Ser-211 or Ser-203. Coimmunoprecipitation revealed an interaction, suggesting that IFI16 modulation of GR function is mediated by protein crosstalk. Transfection analysis with GR mutants showed that the ligand-binding domain of GR binds IFI16 and is the target domain for IFI16 regulation. Analysis of human lung sections identified colocalization of GR and IFI16, suggesting a physiologically relevant interaction. We demonstrate that IFI16 is a novel modulator of GR function and show the importance of analyzing variation in Gc sensitivity in humans, using appropriate technology, to drive discovery.

Circadian timing in the lung; a specific role for bronchiolar epithelial cells.

In addition to the core circadian oscillator, located within the suprachiasmatic nucleus, numerous peripheral tissues possess self-sustaining circadian timers. In vivo these are entrained and temporally synchronized by signals conveyed from the core oscillator. In the present study, we examine circadian timing in the lung, determine the cellular localization of core clock proteins in both mouse and human lung tissue, and establish the effects of glucocorticoids (widely used in the treatment of asthma) on the pulmonary clock. Using organotypic lung slices prepared from transgenic mPER2::Luc mice, luciferase levels, which report PER2 expression, were measured over a number of days. We demonstrate a robust circadian rhythm in the mouse lung that is responsive to glucocorticoids. Immunohistochemical techniques were used to localize specific expression of core clock proteins, and the glucocorticoid receptor, to the epithelial cells lining the bronchioles in both mouse and human lung. In the mouse, these were established to be Clara cells. Murine Clara cells retained circadian rhythmicity when grown as a pure population in culture. Furthermore, selective ablation of Clara cells resulted in the loss of circadian rhythm in lung slices, demonstrating the importance of this cell type in maintaining overall pulmonary circadian rhythmicity. In summary, we demonstrate that Clara cells are critical for maintaining coherent circadian oscillations in lung tissue. Their coexpression of the glucocorticoid receptor and core clock components establishes them as a likely interface between humoral suprachiasmatic nucleus output and circadian lung physiology.

Real-time analysis of gene regulation by glucocorticoid hormones.

There is increasing evidence that temporal factors are important in allowing cells to gain additional information from external factors, such as hormones and cytokines. We sought to discover how cell responses to glucocorticoids develop over time, and how the response kinetics vary according to ligand structure and concentration, and hence have developed a continuous gene transcription measurement system, based on an interleukin-6 (IL-6) luciferase reporter gene. We measured the time to maximal response, maximal response and integrated response, and have compared these results with a conventional, end point glucocorticoid bioassay. We studied natural glucocorticoids (corticosterone and cortisol), synthetic glucocorticoids (dexamethasone) and glucocorticoid precursors with weak, or absent bioactivity. We found a close correlation between half maximal effective concentration (EC50) for maximal response, and for integrated response, but with consistently higher EC50 for the latter. There was no relation between the concentration of ligand and the time to maximal response. A comparison between conventional end point assays and real-time measurement showed similar effects for dexamethasone and hydrocortisone, with a less effective inhibition of IL-6 seen with corticosterone. We profiled the activity of precursor steroids, and found pregnenolone, progesterone, 21-hydroxyprogesterone and 17-hydroxyprogesterone all to be ineffective in the real-time assay, but in contrast, progesterone and 21-hydroxyprogesterone showed an IL-6 inhibitory activity in the end point assay. Taken together, our data show how ligand concentration can alter the amplitude of glucocorticoid response, and also that a comparison between real-time and end point assays reveals an unexpected diversity of the function of glucocorticoid precursor steroids, with implications for human disorders associated with their overproduction.

Glucocorticoid receptor overexpression exerts an antisurvival effect on human small cell lung cancer cells.

Small cell lung cancer (SCLC) is an aggressive tumour with an abysmal prognosis. These cancers are characteristically resistant to glucocorticoid (Gc) action, owing to impaired expression of the glucocorticoid receptor (GR). We identified reduced GR expression in human SCLC cell lines, compared to a non-SCLC cell line. The SCLC cells also showed no Gc inhibition of proliferation, in contrast to non-SCLC cells. Retroviral overexpression of GR resulted in significantly increased cell death, which was partially blocked by the GR antagonist, RU486. Indeed, in cells sorted for GR expression, there was rapid, near complete loss of live cells by 72 h, in contrast to control cells that proliferated as expected. Flow cytometry using Annexin V revealed that cell death was by apoptosis. In addition, confocal analysis of fixed cells showed that cells overexpressing GR displayed a significant increase in fragmenting apoptotic nuclei. Microarray studies showed that transgenic GR expression upregulated the proapoptotic genes, BAD and BAX. We have, therefore, identified a profound apoptotic effect of GR in SCLC cells, which may explain the low levels of endogenous GR in SCLC cells. Understanding how GR overexpression leads to apoptotic cell death in SCLC cells may uncover new therapeutic strategies.

A heterozygous c-Maf transactivation domain mutation causes congenital cataract and enhances target gene activation.

MAF, one of a family of large Maf bZIP transcription factors, is mutated in human developmental ocular disorders that include congenital cataract, microcornea, coloboma and anterior segment dysgenesis. Expressed early in the developing lens vesicle, it is central to regulation of lens crystallin gene expression. We report a semi-dominant mouse c-Maf mutation recovered after ENU mutatgenesis which results in the substitution, D90V, at a highly conserved residue within the N-terminal 35 amino-acid minimal transactivation domain (MTD). Unlike null and loss-of-function c-Maf mutations, which cause severe runting and renal abnormalities, the phenotype caused by the D90V mutation is isolated cataract. In reporter assays, D90V results in increased promoter activation, a situation similar to MTD mutations of NRL that also cause human disease. In contrast to wild-type protein, the c-Maf D90V mutant protein is not inhibited by protein kinase A-dependent pathways. The MTD of large Maf proteins has been shown to interact with the transcriptional co-activator p300 and we demonstrate that c-Maf D90V enhances p300 recruitment in a cell-type dependent manner. We observed the same for the pathogenic human NRL MTD mutation S50T, which suggests a common mechanism of action.

Ovarian Leydig cell tumor in a peri-menopausal woman with severe hyperandrogenism and virilization.

The authors report a case of Leydig cell tumor in a 46-year-old woman who first presented with severe clinical hyperandrogenism and associated complex medical history. Investigations revealed markedly raised serum concentrations of testosterone (28.3 nmol/l) and free androgen index (54.4), whereas sex hormone binding globulin, random cortisol, androstenedione, 17-hydroxyprogesterone and dehydroepiandrosterone sulphate concentrations were all within the normal range. Transabdominal ultrasound and computed tomography scan of the pelvis and abdomen showed a slightly bulky right ovary, but no other abnormalities. An ovarian source of androgens was suspected and surgery was arranged. Following a three-year history of defaulting appointments due to agoraphobia, she underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy and intraoperative selective ovarian venous sampling. Histopathological examination revealed a 2 cm Leydig cell tumor within the right ovary. Successful intraoperative ovarian venous sampling demonstrated significantly elevated testosterone levels (>260 nmol/l) from the right ovarian vein. Hyperandrogenaemia normalized post-operatively. The patient showed significant regression of clinical signs and symptoms, including the anxiety disorder. Clinical presentation, biochemistry and imaging modalities should allow to detect androgen-secreting ovarian tumors, while selective venous sampling should be reserved for patients whom uncertainty remains. The present case confirms that androgen-secreting ovarian tumors represent a diagnostic and therapeutic challenge. They have to be considered in the differential diagnosis of severe hyperandrogenism even in peri-menopausal women. Although selective venous sampling is of diagnostic value, however, its impact on future management should be considered on individual basis.

Glucocorticoids suppress macrophage migration inhibitory factor (MIF) expression in a cell-type-specific manner.

MIF is a potent proinflammatory cytokine involved in inflammatory arthritis. Glucocorticoids (GC) have been reported to induce secretion of MIF in rodent cells, and as MIF counteracts the anti-inflammatory effects of GC, this has implications for human inflammatory disease. Transient transfection studies showed that the MIF promoter was repressed by dexamethasone (Dex) (10 nM) in CEM C7A cells, with up to 50% suppression by 100 nM. However, there was no regulation of the promoter by GC in A549 cells. We also found that subnanomolar concentrations of Dex suppressed MIF secretion, measured by ELISA, by 80% in both human T lymphoblasts (CEM C7A) and human lung epithelial cells (A549). Endogenous MIF mRNA was also repressed by GC in CEM C7A cells, measured both by Northern blot and quantitative RT-PCR assays, but there was no such regulation in A549 cells. This suggests that GC affects translation rather than transcription of MIF in A549 cells. These results contradict earlier results with the rat cell line RAW 264.7. Therefore, we analysed MIF secretion from RAW 264.7 cells but found no GC effect on secretion. Understanding how GC regulates MIF in a cell-type-dependent manner may give insights into GC-refractory human inflammatory diseases.

Analysis of co-factor function in a glucocorticoid-resistant small cell carcinoma cell line.

Human small cell lung carcinoma (SCLC) tumours exhibit neuroendocrine differentiation, secreting hormones such as ACTH and related peptides. While glucocorticoids inhibit ACTH secretion from the pituitary, this does not occur in SCLC tumours and SCLC cell lines. Failure of glucocorticoids to suppress ACTH peptides is accompanied by a global lack of glucocorticoid action in a number of SCLC cell lines. In the human SCLC cell line, COR L103, activation of a human tyrosine aminotransferase (TAT3)-luciferase reporter gene is resistant to glucocorticoids despite similar glucocorticoid receptor (GR) expression to the glucocorticoid-sensitive A549 human lung cancer cell line; moreover, the GR is free of deleterious mutations. Over-expression of a wild-type GR restores glucocorticoid regulation of TAT3-luciferase, and this is enhanced when the activation function (AF)-2 domain is deleted but much reduced when the AF-1 domain is deleted. This suggests aberrant AF-2 activation domain function. We identified defective steroid receptor co-activator 1 (SRC1) recruitment to the GR AF-2 in COR L103 cells, although SRC1 was successfully recruited to the steroid X receptor with rifampicin, suggesting a defect in the GR. Analysis of other GR C-terminal co-factors identified increased expression of nuclear co-repressor (NCoR) in COR L103 cells. To determine the impact of this, NCoR was over-expressed in A549 cells, where it reduced GR transactivation by 55%. In summary, glucocorticoid resistance is associated with altered SRC protein recruitment and increased expression of NCoR in these SCLC cells, suggesting that glucocorticoid sensitivity may be modified by subtle changes in co-factor recruitment.

Macrophage migration inhibitory factor: molecular, cellular and genetic aspects of a key neuroendocrine molecule.

The immunological and neuroendocrine properties of macrophage migration inhibitory factor (MIF) are diverse. In this article we review the known cellular, molecular and genetic properties of MIF that place it as a key regulatory cytokine, acting within both the innate and adaptive immune responses. The unexpected and paradoxical induction of MIF secretion by low concentrations of glucocorticoids is explored. The role of MIF as a locally acting modulator of glucocorticoid sensitivity within foci of inflammation is also discussed. MIF has no homology with any other pro-inflammatory cytokine and until recently lacked a recognised transmembrane receptor. MIF has also been shown to be directly taken up into target cells and to interact with intracellular signalling molecules, including the Jun activation domain-binding protein Jab-1.Comprehensive analysis of the MIF gene has identified important functional polymorphisms and a series of genetic studies has revealed both association and linkage of MIF with inflammatory diseases. Altered MIF regulation may therefore be pivotal to acquiring chronic inflammation following an innate immune response.

Genetic engineering of the glucocorticoid receptor by fusion with the herpes viral protein VP22 causes selective loss of transactivation.

The development of methods for engineering proteins with novel properties opens the way to manipulating intracellular processes in a therapeutically useful way. Glucocorticoids, acting via glucocorticoid receptors (GR), are potent anti-inflammatory agents, acting to oppose nuclear factor kappa B (NF kappa B) function. The herpes viral protein, VP22, has been reported to confer intercellular trafficking activity on 'cargo' proteins, potentially facilitating gene therapy with intracellular proteins. VP22GR, resulting from the addition of VP22 to the N terminal of GR, was equipotent with the wild-type GR in opposing NF kappa B p65-driven expression of an NF kappa B reporter gene. Surprisingly, VP22GR was incapable of inducing transactivation of positive glucocorticoid reporter genes (MMTV-luc and TAT3-luc). Furthermore, the VP22GR had powerful dominant negative activity on both endogenous and exogenous GR transactivation. VP22GR was cytoplasmic in quiescent cells, and after hormone addition underwent nuclear translocation to share the same distribution as the GR. The ability of the VP22GR to selectively confer and enhance glucocorticoid-dependent transrepression of NF kappa B may be of use therapeutically in e.g. transplant rejection, inflammatory arthritis or asthma.

Failure of steroid regulation of the MMTV promoter in a small cell lung cancer cell line is caused by a DNA sequence flanking the glucocorticoid response element.

We have previously described a panel of human small cell lung carcinoma (SCLC) cell lines that have profound glucocorticoid resistance, resulting from various molecular defects in glucocorticoid signalling. However, in one SCLC cell line, CORL103, the cause of the resistance is unknown. These cells are refractory to dexamethasone stimulation of MMTV even when exogenous wild-type glucocorticoid receptor (GR) is co-transfected. This is in contrast to cell lines DMS79 and CORL24 where resistance is overcome by transfection of the wild-type receptor. Sequencing of the GR from CORL103 cells revealed two point mutations, but neither of these induced dominant negative activity. Steroid hormone resistance extended to mineralocorticoid and progesterone receptor (MR, PR) activation of MMTV-luc, whereas oestrogen and thyroid hormone receptor transactivation were normal. A simpler reporter, TAT3-luc, containing three copies of the tyrosine aminotransferase glucocorticoid response element (GRE), was responsive when transfected into CORL103 cells with GR, MR and PR expression vectors and activated with their respective ligands. Similarly, pHH-luc and pAH-luc (truncated MMTV variants containing the GRE region, both derived from a different strain of MMTV), were effectively transactivated with dexamethasone. This suggests that the minor changes in the flanking sequence of the MMTV promoter are critically important in determining steroid responsiveness in CORL103 cells. We propose that minor differences in MMTV may determine recruitment of co-factors, which destabilise GR binding to the MMTV GREs. These findings represent a new, selective, model of glucocorticoid resistance that may explain specific cell and target gene differences in glucocorticoid sensitivity.

Cushing's syndrome due to phaeochromocytoma secreting the precursors of adrenocorticotropin.

Adrenal phaeochromocytoma rarely causes ectopic ACTH syndrome. We describe a 44-yr-old hypertensive woman who was Cushingoid and markedly pigmented. Laboratory studies indicated severe hypokalaemia, abnormal liver function tests, and random serum cortisols greater than 1660 nmol/L. Urinary catecholamines were markedly increased. An abdominal computed tomography scan showed a 4-cm left adrenal mass and an hypertrophied right adrenal. ACTH levels were elevated at 200 pmol/L, but ACTH precursors, which cross-react in the ACTH assay, were more highly elevated at 1625 pmol/L. The tumor cells cultured in vitro also secreted ACTH precursors, whereas ACTH levels were undetectable. Because the patient was highly pigmented, we measured circulating concentrations of alpha-MSH, which were undetectable and certainly insufficient to stimulate melanogenesis, suggesting that tumorderived ACTH precursors or ACTH were responsible for the pigmentation. A laparoscopic adrenalectomy resulted in remission of the Cushing's syndrome and dramatic reduction in the pigmentation. Before operation, treatment of the patient with metyrapone and replacement dexamethasone decreased cortisol from more than 1660 to less than 20 nmol/L. Surprisingly, this resulted in a decrease in ACTH precursors to 100 pmol/L and ACTH to 9.0 pmol/L. In vitro treatment of the tumor cells with dexamethasone for 24 or 40 h increased ACTH precursor secretion. In summary, this phaeochromocytoma causing Cushing's syndrome secreted primarily ACTH precursors, which seemed to cause the marked pigmentation. In vivo and in vitro evidence suggests that glucocorticoids induced ACTH precursor secretion.

Structure/function of the human glucocorticoid receptor: tyrosine 735 is important for transactivation.

Ligand-induced activation of the glucocorticoid receptor (GR) is not well understood. The GR ligand-binding domain was modeled, based on homology with the progesterone receptor. Tyrosine 735 interacts with the D ring of dexamethasone, and substitution of D ring functional groups results in partial agonist steroids with reduced ability to direct transactivation. Loss of the Tyr735 hydroxyl group by substitution to phenylalanine (Tyr735Phe) did not reduce ligand binding affinity [dissociation constant (Kd) 4.3 nM compared with Kd 4.6 nM for wild-type] and did not alter transrepression of an nuclear factor-kappaB (NF-kappaB reporter. But, there was a significant 30% reduction in maximal transactivation of a mouse mammary tumor virus (MMTV) reporter, although with an unchanged EC50 (8.6 nM compared with 6 nM). Substitution to a nonaromatic hydrophobic amino acid, valine (Tyr735Val), retained high-affinity ligand binding for dexamethasone (Kd 6 nM compared with 4.6 nM) and did not alter transrepression of NF-kappaB. However, there was a 36% reduction in MMTV activity with a right shift in EC50 (14.8 nM). The change to serine, a small polar amino acid (Tyr735Ser), caused significantly lower affinity for dexamethasone (10.4 nM). Maximal transrepression of NF-kappaB was unaltered, but the IC50 for this effect was increased. Tyr735Ser had a major shift in EC50 (118 nM) for transactivation of an MMTV reporter. Maximal transactivation of MMTV induced by the natural ligand cortisol was reduced to 60% by Tyr735Phe and Tyr735Val and was completely absent by Tyr735Ser. These data suggest that tyrosine 735 is important for ligand interpretation and transactivation.

The human prolactin gene upstream promoter is regulated in lymphoid cells by activators of T-cells and by cAMP.

Prolactin (PRL) is produced in human thymocytes, T-cells and endometrium. In these extrapituitary tissues, PRL gene transcription is directed by an alternative upstream promoter, and it is thought to act as a locally produced cytokine, with relevance for immune regulation and modulation of T-cell function. We have studied PRL transcriptional regulation in the human T-lymphoblastoid Jurkat cell line transfected with a fragment of the upstream promoter linked to luciferase. A cAMP analogue (cptAMP) increased promoter activity rapidly and dose dependently. This increase was resistant to inhibition by cyclosporin A and thus independent of calcineurin phosphatase (CN). T-cell activation by phorbol myristate acetate (PMA) failed to enhance promoter activity but phytohaemagglutinin (PHA) alone or PHA+PMA increased it, and cptAMP acted in synergy with PMA or PHA to increase it further. H-89, a cAMP-dependent protein kinase A (PKA) inhibitor, inhibited the effect of cptAMP as did transfection with protein kinase inhibitor PKI, an expression vector of the specific inhibitor of PKA. A single point mutation in the CRE (cAMP response element) located at -25 bp in the PRL upstream promoter (TGACGT to TGCCGT) failed to reduce the response to cptAMP, while mutations or deletion of four nucleotides in the CRE to TACTCT diminished the response to cAMP by more than half. We conclude that activity of the human PRL upstream extrapituitary promoter can be induced by activators of T-cells, as well as by a cAMP analogue. The signal is transmitted by PKA and the effect of cAMP is independent of CN. It is partly dependent on an intact proximal CRE motif but a more upstream enhancer may contribute to promoter regulation.

Leukemia inhibitory factor as an intrapituitary mediator of ACTH secretion.

In the normal anterior pituitary, intercellular interactions are important for the expression of hormones including adrenocorticotropin (ACTH). Leukemia inhibitory factor (LIF) is secreted by anterior pituitary cells and stimulates both basal and corticotropin-releasing hormone (CRH)-stimulated ACTH secretion in AtT20 cells. To determine the effects of LIF on normal pituitary cells, we measured the effects of LIF and an immunoneutralizing antiserum against LIF on ACTH secretion by cultured sheep anterior pituitary cells. In intact populations of anterior pituitary cells, LIF (10 nM) stimulated ACTH secretion (from 0.30 +/- 0.06 to 0.77 +/- 0.01 ng/well per 3 h) and antiserum to LIF (by itself) had no effect (0.29 +/- 0.05 ng/well per 3 h). In marked contrast, following pharmacological elimination of CRH-target cells, a condition known to disinhibit ACTH secretion, basal ACTH secretion was elevated (0.74 +/- 0.13 ng/well per 3 h); LIF produced no further stimulation (0.73 +/- 0.22 ng/well per 3 h) but immunoneutralization of LIF significantly reduced secretion to 0.50 +/- 0.10 ng/well per 3 h. Medium, conditioned by exposure to CRH-target-depleted cultures of anterior pituitary cells, increased net ACTH secretion (from 0.29 +/- 0.03 to 6.54 +/- 0.71 ng/well per 3 h), when added as a challenge to naive, cultured anterior pituitary cells. Inclusion of antiserum to LIF significantly attenuated (5.29 +/- 0.62 ng/well per 3 h) this response. The presence in and secretion of LIF by normal individual pituitary cells was detected using immunocytochemical methods. Seven to 8% of all cells stained positively for LIF, with 66 +/- 11% of those secreting detectable amounts of LIF under unstimulated conditions. LIF colocalized with TSH and LH in pituitary cells. Taken together, these data suggest that LIF can stimulate ACTH secretion by normal anterior pituitary cells, and potentially plays a role as an intrapituitary stimulator of ACTH secretion under certain conditions.