The risk of concurrent malignancies in patients with multiple endocrine neoplasia type 1: insights into clinical characteristics of those with multiple endocrine neoplasia type 1.

OBJECTIVE: Summarize and analyze the characteristics of patients with Multiple Endocrine Neoplasia type 1 (MEN-1) who were diagnosed with malignant tumors that do not belong to MEN-1 components. METHODS: Clinical data from patients with MEN-1 who visited Peking Union Medical College Hospital between April 2012 and April 2022 were collected. We compared the clinical characteristics of patients with malignant tumors outside of their MEN-1 components to those without additional tumors. MEN-1 gene testing was performed on most of these patients using Sanger sequencing, whole-exome sequencing, or MLPA. RESULTS: A total of 221 MEN-1 patients were diagnosed, of which 23 (10.40%) were found to have malignant tumors that did not belong to MEN-1 components, including papillary thyroid carcinoma (PTC) (4.52%), breast cancer (1.81%), urologic neoplasms (1.35%), primary hepatic carcinoma (PCC) (0.09%), meningeal sarcoma (0.05%), glioblastoma (0.05%), cervical cancer (0.05%), and lung carcinoma (0.05%). MEN-1 gene mutations were identified in 11 patients, including missense mutations, frameshift mutations, and splice mutations. The prevalence of each endocrine neoplasm, particularly gastroenteropancreatic neuroendocrine tumor, was higher in MEN-1 patients with other malignant tumors compared to MEN-1 patients without malignant tumors. CONCLUSION: Our retrospective study revealed a higher incidence of non-MEN-1 component malignant tumors in MEN-1 patients, especially breast cancer, PTC, and urologic neoplasms. These patients also exhibit more severe clinical phenotypes of MEN-1.

Concurrent mutations of germline GPR101 and somatic USP8 in a pediatric giant pituitary ACTH adenoma: a case report.

BACKGROUND: Cushing's disease (CD) is rare in pediatric patients. It is characterized by elevated plasma adrenocorticotropic hormone (ACTH) from pituitary adenomas, with damage to multiple systems and development. In recent years, genetic studies have shed light on the etiology and several mutations have been identified in patients with CD. CASE PRESENTATION: A girl presented at the age of 10 years and 9 months with facial plethora, hirsutism and acne. Her vision and eye movements were impaired. A quick weight gain and slow growth were also observed. Physical examination revealed central obesity, moon face, buffalo hump, supra-clavicular fat pads and bruising. Her plasma ACTH level ranged between 118 and 151 pg/ml, and sella enhanced MRI showed a giant pituitary tumor of 51.8 × 29.3 × 14.0 mm. Transsphenoidal pituitary debulk adenomectomy was performed and immunohistochemical staining confirmed an ACTH-secreting adenoma. Genetic analysis identified a novel germline GPR101 (p.G169R) and a somatic USP8 (p. S719del) mutation. They were hypothesized to impact tumor growth and function, respectively. CONCLUSIONS: We reported a rare case of pediatric giant pituitary ACTH adenoma and pointed out that unusual concurrent mutations might contribute to its early onset and large volume.

The Diverse Analysis Identifies Mutated KRAS Associated With Radioresistance in Non-Small Cell Lung Cancer.

Background: To analyze the relationship between V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) status and radioresistance in non-small cell lung cancer (NSCLC), we identified potential genotypic differences and pathways involved. Methods: We retrospectively analyzed epidermal growth factor receptor (EGFR) and KRAS status in patients undergoing definitive radiotherapy for NSCLC between 2004 and 2018. Cox proportional hazard models were used to evaluate local progression-free survival (LPFS). Using clonogenic survival and measurement of γH2AX foci, we analyzed the difference in radiosensitivity between NSCLC cell lines with different KRAS status. The Cancer Genome Atlas (TCGA) analysis was used to explore the potential pathways involved. Results: The results showed that of the 286 patients identified, 68 (24%) had local tumor progression (mean ± standard deviation (SD), 27 ± 17.4 months); of these patients, KRAS mutations were found in 14 (23%), and KRAS status was associated with LPFS. After adjusting for concurrent chemotherapy, gross tumor volume, and mutation status in multivariate analysis, KRAS mutation was associated with shorter LPFS (hazard ratio: 1.961; 95% confidence interval: 1.03 - 2.17; P = 0.032). KRAS mutation showed higher radioresistance in vitro. TCGA data showed that the ERK1/2 pathway, phosphatidylinositol I3 kinase (PI3K)/mTOR, p38 MAPK pathway, cell cycle checkpoint signaling, DNA damage, repair pathways, and EGFR/PKC/AKT pathway were differentially expressed in patients with KRAS mutations or cell lines compared with their expression in the wild-type group. Conclusions: Diverse analyses identified that KRAS mutation was associated with radioresistance in NSCLC. KRAS mutation status may be helpful as a biomarker of radioresistance and a potential target to increase radiosensitivity.

Appendiceal Neuroendocrine Tumor Is a Rare Cause of Ectopic Adrenocorticotropic Hormone Syndrome With Cyclic Hypercortisolism: A Case Report and Literature Review.

OBJECTIVE: Ectopic adrenocorticotropic hormone (ACTH) syndrome (EAS) is a condition of hypercortisolism caused by non-pituitary tumors secreting ACTH. Appendiceal neuroendocrine tumor as a rare cause of ectopic ACTH syndrome was reported scarcely. We aimed to report a patient diagnosed with EAS caused by an appendiceal neuroendocrine tumor and summarized characteristics of these similar cases reported before. CASE REPORT AND LITERATURE REVIEW: We reported a case with Cushing's syndrome who was misdiagnosed as pituitary ACTH adenoma at first and accepted sella exploration. Serum and urinary cortisol decreased, and symptoms were relieved in the following 4 months after surgery but recurred 6 months after surgery. The abnormal rhythm of plasma cortisol and ACTH presented periodic secretion and seemingly rose significantly after food intake. EAS was diagnosed according to inferior petrosal sinus sampling (IPSS). Appendiceal mass was identified by 68Ga-DOTA-Tyr3-octreotate (DOTATATE)-PET-CT and removed. The pathological result was consistent with appendiceal neuroendocrine tumor with ACTH (+). The literature review demonstrated 7 cases diagnosed with EAS caused by appendiceal neuroendocrine tumor with similarities and differences. CONCLUSION: The diagnosis of an ectopic ACTH-producing tumor caused by the appendiceal neuroendocrine tumor can be a challenging procedure. Periodic ACTH and cortisol secretion may lead to missed diagnosis and misdiagnosis. IPSS is crucial in the diagnosis of EAS and 68Ga-DOTATATE-PET-CT plays an important role in the identification of lesions.

Clinical characteristics of non-alcoholic fatty liver disease in Chinese adult hypopituitary patients.

BACKGROUND: Patients with hypothalamic-pituitary disease have the feature of central obesity, insulin resistance, and dyslipidemia, and there is increased prevalence of liver dysfunction consistent with non-alcoholic fatty liver disease (NAFLD) in this population. The causes of hypopituitarism in the reported studies varied and combined pituitary hormone deficiency including central diabetes insipidus is much common in this population. This retrospective cross-sectional study was performed to analyze the clinical characteristics and related factors with NAFLD and cirrhosis in Chinese adult hypopituitary/panhypopituitary patients. AIM: To analyze the clinical characteristics of and related risk factors for NAFLD in Chinese adult hypopituitary patients. METHODS: Adult Chinese patients with hypopituitarism and/or panhypopituitarism were enrolled at the Pituitary Center of Peking Union Medical College Hospital between August 2012 and April 2018. According to abdominal ultrasonography, these patients were divided into an NAFLD (-) group and an NAFLD (+) group, and the latter was further divided into an NAFLD group and a cirrhotic group. The data, such as patient characteristics, diagnosis, and treatment, were extracted from medical records, and statistical analysis was performed. RESULTS: A total of 36 male and 14 female adult Chinese patients with hypopituitarism were included in this retrospective study; 43 (87.0%) of these patients exhibited growth hormone (GH) deficiency, and 39 (78.3%) had diabetes insipidus. A total of 27 (54.0%) patients were diagnosed with NAFLD, while seven patients were cirrhotic. No significant differences were noted in serum GH or insulin-like growth factor 1 among patients with cirrhosis, subjects with NAFLD, and those without NAFLD. However, plasma osmolality and serum sodium concentration of the cirrhotic patients were 314.9 mOsm/kgH2O and 151.0 mmol/L, respectively, which were significantly higher than those of the NAFLD patients (P = 0.036 and 0.042, respectively). Overweight/obesity and insulin resistance were common metabolic disorders in this population. The body mass index (BMI) and homeostasis model assessment of insulin resistance parameters of the cirrhotic patients were 27.7 kg/m2 and 9.57, respectively, which were significantly higher than those of the patients without NAFLD (P = 0.011 and 0.044, respectively). A correlation analysis was performed, and fasting insulin concentration was positively associated with plasma osmolality in patients with NAFLD, after adjusting for gender, age, and BMI (r = 0.540, P = 0.046), but no correlation was noted in patients without NAFLD. CONCLUSION: NAFLD is common in patients with hypopituitarism. Plasma osmolality and serum sodium levels of hypopituitary patients with cirrhosis are higher than those of subjects with NAFLD, and fasting insulin concentration is positively associated with plasma osmolality in patients with NAFLD, which suggests that hyperosmolality might be a contributor to the worsening of NAFLD in hypopituitary patients.

The changes of serum glypican4 in obese patients with different glucose metabolism status.

CONTEXT AND OBJECTIVE: Glypican 4 (Gpc4) was identified as a novel adipokine capable of intensifying insulin signaling and regulating adipocyte differentiation. This study was performed to investigate the changes of serum Gpc4 levels in obese patients with different glucose metabolism status and its association with metabolic-related parameters. DESIGN AND PARTICIPANTS: A total of 170 obese patients with different glucose metabolism status and 38 normal controls were recruited, and obese patients were divided into 4 groups: OB1, obese patients with normal glucose tolerance (NGT) and normal insulin levels; OB2, obese patients with normal glucose tolerance and hyperinsulinemia; OB3, obese patients with impaired glucose tolerance; and OB4, obese patients with type 2 diabetes mellitus. Serum Gpc4 was determined by commercially available ELISA kits. RESULTS: Serum Gpc4 levels in the OB2, -3, and -4 groups were significantly increased in comparison with that in the OB1 group (3.5 [2.0-5.3] ng/mL, 3.0 [1.5-6.1] ng/mL, and 3.4 [1.8-5.4] ng/mL vs 1.9 [1.3-4.3] ng/mL, P < .05). The levels were positively correlated with body mass index (BMI), systolic blood pressure (SBP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), fasting insulin (FINS), and homeostasis model assessment of insulin resistance (HOMA-IR) in all subjects. Multiple linear regression showed that SBP, AST, HOMA-IR, and FINS were independent contributors to circulating Gpc4 levels after adjusting for age, gender, and BMI in all subjects (P < .05). Additionally, serum Gpc4 levels in males of the normal group and the OB3 group were higher than those in females (2.9 [2.1-4.9] ng/mL vs 1.6 [1.2-3.1] ng/mL; 4.8 [2.5-6.3] ng/mL vs 2.7 [1.1-4.4] ng/mL, P < .05). CONCLUSIONS: Serum Gpc4 levels were significantly elevated in obese patients with insulin resistance and positively correlated with BMI, SBP, ALT, AST, FINS, and HOMA-IR, suggesting Gpc4 is a novel adipokine associated with obesity and insulin resistance-related metabolic disorders.

Serum Levels of the Adipokine Zinc- α 2-glycoprotein Are Decreased in Patients with Hypertension.

Objective. Zinc-α2-glycoprotein (ZAG) has recently been proposed as a new adipokine involved in body weight regulation. The purpose of this study is to investigate serum levels of ZAG in patients with hypertension and its association with related characteristics. Methods. 32 hypertension patients and 42 normal controls were recruited and the relationship between serum ZAG, total and high molecular weight (HMW) adiponectin, and tumor necrosis factor-α (TNFα) determined by enzyme-linked immunosorbent assay (ELISA) and metabolic-related parameters was investigated. Results. Serum ZAG concentrations were significantly lowered in patients with hypertension compared with healthy controls (61.4 ± 32 versus 78.3 ± 42 µg/mL, P < 0.05). The further statistical analysis demonstrated that serum ZAG levels were negatively correlated with waist-to-hip ratio (WHR) (r = -0.241, P < 0.05) and alanine aminotransferase (ALT) (r = -0.243, P < 0.05). Additionally, serum HMW adiponectin significantly decreased, while TNFα greatly increased in hypertension patients as compared with healthy controls (2.32 ± 0.41 versus 5.24 ± 1.02 µg/mL, 3.30 ± 1.56 versus 2.34 ± 0.99 pg/mL, P < 0.05). Conclusions. Serum ZAG levels are significantly lowered in hypertension patients and negatively correlated with obesity-related item WHR, suggesting ZAG is a factor associated with hypertension.

Inhibition of preadipocyte differentiation and adipogenesis by zinc-α2-glycoprotein treatment in 3T3-L1 cells.

AIMS/INTRODUCTION: Zinc-α2-glycoprotein (ZAG) is associated with the loss of adipose tissue in cancer cachexia, and has recently been proposed to be a candidate factor in the regulation of bodyweight. The aim of the study was to investigate the effects of ZAG on the proliferation and differentiation of 3T3-L1 preadipocytes. MATERIALS AND METHODS: 3-(4,5-Dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) spectrophotometry, Oil Red O staining, intracellular triglyceride assays, real-time quantitative reverse transcription polymerase chain reaction and transient transfection methods were used to explore the action of ZAG. RESULTS: Ectopic ZAG expression significantly stimulates 3T3-L1 cells proliferation in a dose- and time-dependent manner. The maximum influence of ZAG on proliferation was 1.43-fold higher than what was observed in control cells. This effect was observed 144 h after transfection with 0.16 µg of murine ZAG (mZAG) plasmid (P < 0.001). The intracellular lipids content in mZAG over-expressing cells were decreased as much as 37% when compared with the control cells after differentiation (P < 0.05, P < 0.01). The messenger ribonucleic acid levels of peroxisome proliferators-activated receptor-γ (PPARγ), CCAAT enhancer-binding protein-α (C/EBPα) and the critical lipogenic gene, fatty acid synthase (FAS), are also downregulated by up to 50% in fully differentiated ZAG-treated adipocytes. ZAG suppresses FAS messenger ribonucleic acid expression by reducing FAS promoter activity. CONCLUSIONS: Zinc-α2-glycoprotein stimulates the proliferation and inhibits the differentiation of 3T3-L1 murine preadipocytes. The inhibitory action of ZAG on cell differentiation might be a result of the attenuation of the expression of PPARγ, C/EBPα and the lipogenic-specific enzyme FAS by reducing FAS promoter activity.

Generalized glucocorticoid resistance accompanied with an adrenocortical adenoma and caused by a novel point mutation of human glucocorticoid receptor gene.

BACKGROUND: Generalized glucocorticoid resistance syndrome is a rare familial or sporadic condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. This syndrome is partially caused by mutations in the human glucocorticoid receptor (hGR) gene. The clinical spectrum of generalized glucocorticoid resistance is broad, ranging from fatigue or no symptoms to severe hypertension with hypokalemic alkalosis. The purpose of this study was to explore the genetic disorder of glucocorticoid resistance syndrome. METHODS: We identified a 56-year-old male patient diagnosed with generalized glucocorticoid resistance syndrome accompanied with an adrenocortical adenoma. This asymptomatic patient referred to Peking Union Medical College Hospital for treatment of his adrenal incidentaloma. Endocrinological evaluation consistently revealed his elevated serum cortisol level. Total RNA was extracted from the patient's peripheral blood mononuclear leukocytes (PBMLs) and entire coding region of hGR alpha was amplified by reverse transcription (RT)-PCR. To confirm the possible mutation identified by sequencing RT-PCR products, genomic DNA sequence of hGR gene from the patient and 50 healthy controls was analyzed by PCR and directly sequencing. RESULTS: A heterozygotic (C→T) substitution at nucleotide position of 1667 (exon 5) in GR alpha gene was found in this patient by sequencing of RT-PCR products of hGR gene. This substitution was also identified at genomic DNA level and it was absent in 100 chromosomes from 50 unrelated health controls. This substitution resulted in a threonine to isoleucine substitution (ACT→ATT) at amino acid 556 in the ligand-binding domain of GR alpha. CONCLUSION: Generalized glucocorticoid resistance in this patient might be caused by a novel heterozygotic mutation in the ligand-binding domain of the GR alpha.

[Activin inhibits the promoter activity of human growth hormone gene in rat pituitary GH3 cells].

The present study was aimed at investigating the effect of activin on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the underlying molecular mechanism. The method of luciferase reporter gene was used. We firstly established a stable GH3 cell line which contains hGH gene promoter (-484 to 30 bp) and luciferase reporter gene by transfecting pGL3-484-Luc2 luciferase expression plasmid into GH3 cells using Lipofectamine transfection reagent. After treating these cells with activin or activin plus various signaling transduction activators, the concentration of GH in the medium and lysate of GH3 cells and luciferase activities in GH3 cells were measured. The results showed that activin (5 nmol/L, 50 nmol/L) decreased the secretion and synthesis of GH. The amounts of GH content in GH3 lysate and medium treated with 50 nmol/L activin were 82% and 59% of the control, respectively. Furthermore, activin (5, 50 nmol/L) reduced the luciferase expression in stable GH3 cells, with the expression being 77% and 69% of the control (P<0.001). Among the activators of intracellular signaling transduction pathways, mitogen-activated protein kinases kinase (MAPKK/MEK) activators C(6) ceramide (1 micromol/L) abolished completely the inhibitory effect of activin. Western blot analysis further confirmed the inhibition of phosphorylated MEK in GH3 cells. The inhibitory effect of activin was abrogated following the deletion of the fragment from -132 to -66 bp within the hGH gene promoter. These results indicate that activin decreases the activity of hGH gene promoter in rat pituitary GH3 cells. The intracellular MEK dependent signaling pathway and the promoter sequence that spans the -132 to -66 bp fragment of hGH gene are involved in the inhibitory effect of activin.

Mek and p38 MAPK-dependant pathways are involoved in the positive effect of interleukin-6 on human growth hormone gene expression in rat MtT/S somatotroph cells.

OBJECTIVE: To investigate the effect of interleukin-6 (IL-6) on the human growth hormone (hGH) gene expression in a rat somatotropic pituitary cell line MtT/S. METHODS: The plasmids containing various lengths of hGH gene 5'-promoter fragments were constructed. Stably transfected MtT/S cells were created by cotransfecting the above plasmids and pcDNA3. 1(+) with DMRIE-C transfection reagent After the administration of these cells with IL-6 and/or various inhibitors of signaling transduction pathways, the luciferase activities in MtT/S cells lysis were assayed to demonstrate the effects of IL-6 on hGH gene promoter activity and possibly involved mechanism. RESULTS: The 10(3) U/mL IL-6 stimulated GH secretion and synthesis, and promoted the 5'-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.69 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 micromol/L) and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 micromol/L) completely blocked the stimulatory effect of IL-6. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected IL-6 induction of hGH promoter activity. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-6. The results showed that the stimulatory effect of IL-6 was abolished following deletion of the -196 to - 132 bp fragment. CONCLUSIONS: IL-6 promotes GH secretion and synthesis by rat MtT/S somatotroph cells. The stimulatory effect of IL-6 on hGH gene promoter appears to require the activation of MEK and p38 MAPK, and a fragment of promoter sequence that spans the - 196 to - 132 bp of the gene, but may be unlinked with Pit-1 protein.

Effect of interleukin-1 beta on growth hormone gene expression and its possible molecular mechanism in rat MtT/S somatotroph cells.

OBJECTIVE: To elucidate the effect of interleukin-1 beta (IL-1 beta) on human growth hormone (hGH) gene expression in a rat somatotropic pituitary cell line MtT/S. METHODS: Stably transfected MtT/S cells were firstly established by transfecting 484-Luc1 plasmid which contained hGH gene promoter -484 to +30 bp and luciferase reporter gene. The effect of IL-1 beta on hGH gene expression was determined by assaying the luciferase activities. RT-PCR method was also used to determine whether IL-1 recepor mRNA was expressed in MtT/S cells. RESULTS: The 10(3) U/mL IL-1 beta stimulated secretion and synthesis of GH, and promoted the 5'-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.38 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 micromol/L) and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 micromol/L) completely blocked the stimulatory effect of IL-1 beta, and phosphatidylinositol-3-kinase (PI3-K) inhibitor LY294002 partly abolished the effect of IL-1 beta. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected induction of hGH promoter activity by IL-1 beta. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-1 beta, and results showed that the stimulatory effect of IL-1 beta was abolished following deletion of the -196 to -132 bp fragment. CONCLUSIONS: IL-1 beta promotes GH secretion and synthesis in rat MtT/S somatotroph cells. The stimulatory effect of IL-1 beta on hGH gene promoter appears to require the activation of MEK, p38 MAPK, PI3-K, and a fragment of promoter sequence that spans the -196 to -132 bp of the gene, but it may be unlinked with Pit-1 protein.

The regulatory mechanism by which interleukin-6 stimulates GH-gene expression in rat GH3 cells.

The present study was performed to elucidate the effect of interleukin (IL)-6 on the human GH (hGH)-gene expression in GH3 rat pituitary tumor cells using stable transfection of the hGH promoter fused to a luciferase reporter gene. Our results showed that IL-6 (10(2)-10(4) U/ml) stimulated GH secretion and synthesis, and promoted the luciferase expression in stably transfected GH3 cells with the maximal action of 1.99 times above the control by 10(4) U/ml IL-6. Among the inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 microM) and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 microM) completely blocked the stimulatory effect of IL-6. Western blot analysis demonstrated that IL-6 indeed increased the activation of phosphorylated MEK and p38 MAPK in GH3 cells. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IL-6 induction of hGH-promoter activity. To identify the DNA sequence that mediated the effect of IL-6, six deletion constructs of hGH promoter were created. The stimulatory effect of IL-6 was abolished following deletion of the -196 to -132 bp fragment. In conclusion, our data show that IL-6 promotes GH secretion and synthesis by rat pituitary GH3 cells. The stimulatory effect of IL-6 on hGH-gene promoter appears to require the activation of MEK and p38 MAPK, and a fragment of promoter sequence that spans the -196 to -132 bp of the gene, but may be unrelated to Pit-1 protein.

Stimulatory effect of interleukin-1beta on growth hormone gene expression and growth hormone release from rat GH3 cells.

Our previous studies demonstrated that interferon gamma increases the human (h) growth hormone (GH) gene promoter activity in rat pituitary GH3 cells, and its regulatory mechanism may be different from the classical GH-releasing hormone-induced regulatory mechanism. Interleukin-1beta (IL-1beta) is thought to induce the release of GH by pituitary cells, but whether or not and by which mechanisms IL-1beta regulates GH synthesis remains unclear. The purpose of our study was thus to investigate the effect of IL-1beta on the hGH gene expression in GH3 rat pituitary tumor cells using stable transfection of the hGH promoter fused to a luciferase reporter gene. Our results showed that IL-1beta (10-10(4) U/ml) increased GH secretion and synthesis and that 10(2) to 10(4) U/ml IL-1beta promoted the luciferase expression in stable GH3 cells, with a maximal action of 1.61 times over that of controls. Among inhibitors of intracellular signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 microM) and p38 MAPK inhibitor SB203580 (5 microM)blocked completely the stimulatory effect of IL-1beta, and the phosphoinositide 3-kinase inhibitor LY294002 (10 microM) blocked partially the induction of IL-1beta. Western blot analysis demonstrated that IL-1beta increased the activation of phosphorylated MEK and p38 MAPK in GH3 cells. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IL-1beta induction of hGH promoter activity. To identify the DNA sequence that mediated the effect of IL-1beta, six deletion constructs of hGH promoter were created. The stimulatory effect of IL-1beta was abolished following deletion of the -196- to -132-bp fragment. In conclusion, our data show that IL-1beta promotes GH secretion and synthesis by rat pituitary GH3 cells. The stimulatory effect of IL-1beta on the hGH gene promoter appears to require the activation of MEK, p38 MAPK, and phosphoinositide 3-kinase and a fragment of promoter sequence that spans the -196- to -132-bp fragment of the gene, but is unrelated to the Pit-1 protein.

IFN-gamma increases the hGH gene promoter activity in rat GH3 cells.

AIM: To study the effect(s) of interferon gamma (IFN-gamma) on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the molecular mechanism underlying the effect(s). METHODS: Cell transfection and luciferase reporter gene were used. RESULTS: IFN-gamma (10(2) and 10(3) U/ml) increased the activity of hGH in GH3 cells. The addition of the mitogen-activated protein kinase inhibitor PD98059 (40 micromol/l) to the cells blocked the stimulatory effect of IFN-gamma. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IFN-gamma induction of hGH promoter activity. To identify the DNA sequence that mediated the effect of IFN-gamma, four deletion constructs of hGH gene promoter were created. The stimulatory effect of IFN-gamma was abolished following deletion of the -250 to -132 fragment. CONCLUSIONS: IFN-gamma increases the activity of hGH gene promoter in rat pituitary GH3 cells. This stimulatory effect of IFN-gamma appears to require the intracellular mitogen-activated protein kinase-dependent signaling pathway. The effect of IFN-gamma requires the promoter sequence that spans the -250 to -132 fragment of the gene, but is unrelated to Pit-1 protein.

[The Effect of Cytokines on Expression of Growth Hormone Gene in Rat Pituitary GH(3) Cells].

To study the effect of interleukin-11(IL-11), ciliary neurotropic factor (CNTF) and transforming growth factor-beta (TGF-beta) on the hGH gene promoter activity in rat pituitary GH(3) cells and the interaction with pituitary-specific transcription factor Pit-1, firstly the stable transformed GH(3) cell line which contained hGH gene promoter 484-30 bp and luciferase reporter gene was established, then the concentration of GH in the medium and lysate of GH(3) cells and luciferase activities in GH(3) cells were measured, after treating these cells with the above cytokines, the effects of cytokines on secretion and synthesis of GH, and the promoter activity of the hGH gene were observed. The results of our experiments showed that IL-11(20 nmol/L), CNTF(10 nmol/L) and TGF-beta(5 nmol/L) regulated secretion and synthesis of GH, and the luciferase expression in stable-transformed GH(3) cells. IL-11 and CNTF had a stimulatory effect, whereas TGF-beta had an inhibitory one. Neither overexpression of Pit-1 nor inhibition of Pit-1 expression could affect the regulatory role of these cytokines. In conclusion, IL-11, CNTF and TGF-beta regulated the GH production in pituitary GH(3) cell line by regulating the hGH gene promoter activity, while Pit-1 might not be involved in the roles.