| BackgroundGrowth hormone is a 191-amino acid, with a molecular weight of 22,124 Daltons, single-chain polypeptide. Genes for human growth hormone are localized in the q22-24 region of chromosome 17. It is synthesized, stored, and secreted in impulse type by somatotropic cells within the lateral wings of the anterior pituitary gland.Growth hormone (GH) is an essential hormone to human body. The major effect of GH is increasing body height during childhood. Besides promoting the postnatal growth and development, bone calcium deposition, it is also engaged in the regulation of fat and energy metabolism and the stimulation of the immune system, and can increase the serum glucose and free fatty acid concentration under stress.[1,2] Growth hormone deficiency (GHD) can cause various diseases, which main manifestations are growth disorders and short stature for children, and metabolic disorders, osteoporosis and even psychological disorder for adults. At present, the only solution to this problem is to supplement exogenous growth hormone.Prior to its production by recombinant DNA technology, treat-used growth hormone was extracted from the pituitary glands of human. Its production was therefore extremely low. It was used strictly and the dosage was hard to satisfy. Until 1985, cadaver growth hormone were forbidden to use because of medication safety, at the same time recombinant human growth hormone (rhGH) was approved by FDA to be used for GHD in children and it quickly replaced pituitary-derived human growth hormone. Since then applications of rhGH was gradually expanded. It was not only used for the treatment for children’s growth disorders caused by GHD such as short stature, penis dysplasia, delayed bone growth, and also the treatment for decreased muscle mass, osteoporosis in adults caused by GHD. It is also used for other diseases independent of GHD such as idiopathic short stature, Turner’s syndrome (TS) in children. In addition, because rhGH can reverse many nutritional and metabolic abnormalities associated with severe catabolic states, it is still being administered adjunctively in patients with chronic renal failure, burns, major surgery, sepsis and HIV/AIDS wasting syndrome, etc.Along with the widespread clinical use of rhGH, kinds of adverse effects have become a problem of drug safety. The adverse effects include drug hypersensitivity, metabolic and nutritional disorders, promotion of liver fibrosis, scoliosis, leukemia risk, insulin resistance (IR), cardiorespiratory compromise and even sudden death. However, the molecular mechanisms of the adverse effects induced by exogenous growth hormone treatment are not clearly known. Therefore, studying the molecular mechanism of growth hormone may help us understand the possible causes of these adverse effects caused by treatment of exogenous growth hormone and further provide helps for the solution to this problem.Growth is simulated by growth hormone at mainly 2 mechanisms. Both start with its binding to the growth hormone receptor (GHR) on target cells, and subsequently activating different signaling pathways. The one is activating the MAPK/ERK pathway after binding to GHR, through which growth hormone directly stimulates division and multiplication of chondrocytes of cartilage. The other one is activating the JAK-STAT signaling pathway after binding to GHR and then producing insulin-like growth factor 1 (IGF-1). IGF-1 has growth-stimulating effects on a wide variety of tissues and can stimulate osteoblast and chondrocyte activity to promote bone growth. In addition, the combination of GH with GHR can activate other signaling pathways such as the RAS-RAF-ERK pathway and the PI3K pathway, etc.Liver is the major target organ of growth hormone and the principal site of IGF-1 production. The JAK-STAT signaling pathway is the major pathway of growth hormone in liver. Thus, this paper selects the JAK-STAT signaling pathway of growth hormone in liver as target spot to study the influences of long-term administration of growth hormone on the JAK-STAT signaling pathway in liver.Signal transduction of JAK-STAT signaling pathway in liver is initiated by ligand binding of GH to the GHR dimer. In the protein structure of GH, there are 4 single-ring structures which could be combined with GHR. After GHR dimer is bound to GH, the intracellular part of GHR then brings two Janus kinase 2 (JAK2) molecules into proximity, which phosphorylates itself and GHR. Phosphorylated GHR could in return activate the phosphorylation of JAK, forming a cross phosphorylation. Together with GH, phosphorylated GHR and JAK2 form a GH-GHR-JAK2 multimer, which can provide docking sites for signaling mediators.[2, 17]Although STAT1, STAT3 and STAT5 of the signal transducers and activators of transcription (STATs) family all can be activated in the JAK-STAT pathway, STAT5 is thought as the major signal molecule.[18] STAT5 consists of two homologous isoforms STAT5a and STAT5b, which are encoded by two juxtaposed genes on mouse chromosome 11 and human chromosome 17.[191 STAT5 is phosphorylated by the GH-GHR-JAK2 multimer after bound to the docking sites for signaling mediators, turned into phosphorylated STAT5 (p-STAT5). Subsequently, p-STAT5 then form a dimer and translocate to the nucleus, where it binds to specific DNA response elements, (usually an inverted repeat of TTCN3GAA), to modulate target gene such as the suppressor of cytokine signaling 2 (SOCS-2) gene and IGF-1 gene to transcript. [19]This paper studies the influence of long-term administration of exogenous growth hormone on liver JAK-STAT signaling pathway from both in vitro and in vivo.ObjectiveLiver is the major target organ of growth hormone and the JAK-STAT pathway is the major signaling pathway of growth hormone in liver. STAT5 is the key effect signal factor of this signaling pathway. This study will test the level of phosphorylation of STAT5 in liver under the condition of long-term administration of growth hormone from both in vivo and in vitro, and further explore the effect of long-term administration of growth hormone on the JAK-STAT signaling pathway in liver.Methods1. Cell culture study:This study selected a human liver cancer cell line Hep G2 and cultured the cells with DMEM which contained 10%(volume fraction) fetal bovine serum. They were cultivated in a CO2 incubator at 37℃ constant temperature.1.1 Hep G2 cells of logarithmic growth phase were evenly passaged into 8 wells of a 12-well cell culture plate and cultured in DMEM which contained 10% fetal bovine serum. Original medium was changed with serum-free DMEM when cell density reached about 90%. After 9 hours of serum starvation, cells were divided into 4 groups:Omin rhGH group, 10min rhGH group,30min rhGH group and 50min rhGH group,2 wells for each group. Each group was treated with rhGH (working concentration of 3000ng/ml) for 50 minutes,30 minutes,10 minutes and 0 minutes separately according to the groups. Each time one group was treated with rhGH, the other three groups were treated with equal volume of serum-free DMEM. Then cells were lysed by RIPA on ice. Lysates were collected to extract cell proteins and to test the level of p-STAT5.1.2 Clean and smooth sterile glass coverslip was placed in each well of a 12-well cell culture plate. Hep G2 cells of logarithmic growth phase were evenly passaged into 6 wells, with cell density of about 30%. Cells were cultured in DMEM which contained 10%fetal bovine serum. Original medium was change with serum-free DMEM when cells firmly adhered to the coverslip. After 9 hours of serum starvation, cells were divided into 3 groups:Omin rhGH group,20min rhGH group,60min rhGH group,2 wells for each group. Then each group was treated with rhGH (working concentration of 3000ng/ml) for 60 minutes,20 minutes and 0 minutes separately according to the groups. Each time one group was treated with rhGH, the other three groups were treated with equal volume of serum-free DMEM. The coverslip s were removed from the wells at normal temperature and the level of p-STAT5 was tested after cells were fixed by 4% paraformaldehyde,1.3 Hep G2 cells of logarithmic growth phase were evenly passaged into 4 wells of a 12-well cell culture plate and cultured in DMEM which contained 10% fetal bovine serum, making the cell density being about 50% when cells attached to the surface of the wells. Original medium was then changed with DMEM containing 2% FBS. Cells were then divided into 2 groups:the long-term GH group and the control group,2 wells for each group. The long-term GH group was treated with rhGH (working concentration:3000ng/ml) daily for a total 3 days. The control group was treated with equal volume of serum-free DMEM each time. Cell density was controlled to be about 90% after 24 hours from the last treatment. Then original medium was changed with serum-free DMEM for a 9-hour-long serum starvation. After that every group was treated with a single dose of rhGH (working concentration: 3000ng/ml).30 minutes later, cells were lysed by RIPA on ice. Cell lysates were collected to extract cellular proteins and to test the level of p-STAT5.2. Animal study:This study selected 12 healthy male BALB/c mice aged 6-8 weeks. Mice were divided into 2 groups randomly:the long-term GH group and the control group,6 mice for each group. The long-term GH group was given an intraperitoneal injection of rhGH at 1 μg/g body weight/time once a day for 3 weeks. The control group was given equal volume of normal saline each time. Sixteen hours later from the last injection, mice were sacrificed on 8:00 in the next day morning. Thirty minutes prior to the sacrifice,3 mice of each group were given a single intraperitoneal injection of rhGH at lug/g body weight, and the other 6 mice were given equal volume of normal saline. Finally,12 mice were divided into 4 groups:the control group, the single dose GH group, the long-term GH group, and the long-term+single dose GH group. Mice were fully anesthetized with chloral hydrate. Livers were quickly resected, frozen in liquid nitrogen, and then stored in a -80 ℃ freezer for further test. Then Liver tissues were ground and lysed with RIPA on ice. Tissue protein was then extracted and the level of p-STAT5 was tested. During the whole experiment, the daily injection time and the sacrifice time shall be ensured to be consistent. Injection was made alternately in two groups each time.3. Determination of protein signaling molecules.3.1 The total cellular protein from experiment 1.1 and 1.3, and the tissue protein from experiment 2 were tested for p-STAT5 level by Western Blot.3.2 Coverslips of Hep G2 were stained by immunofluorescence and then observed for fluorescence intensity of p-STAT5 protein with a fluorescence microscope.4. Statistical AnalysisAll the data were recorded in WPS excel 2015 and analyzed with SPSS 20.0. The results were presented as arithmetic mean±standard deviation (M±SD). Comparisons between two groups were conducted by Independent Sample T Test when variance was homogeneous or Satterthwaite t test when variance was heterogeneous. If P value<0.05, it means there is a significant difference and it owns statistical significance.Results1. Effect of long-term administration of growth hormone on the level of p-STAT5 in Hep G2 cells in vitro study (cell culture study).1.1 The experiment results showed that the level of p-STAT5 of Hep G2 cells of the 50min rhGH group (gray value of 0.55±0.18) was significantly less than that of the l0min rhGH group (gray value of 1.45±0.39) and the 30min rhGH group (gray value of 1.49±0.22) (P=0.023 vs. l0min rhGH group; P=0.005 vs.30min rhGH group). There was no significant difference between the l0min rhGH group and the 30min rhGH group (P=0.879).1.2 The experiment results demonstrated that the fluorescence intensity of p-STAT5 of Hep G2 cells of the 60min rhGH group was obviously weaker than that of the 20min rhGH group.1.3 The experiment results indicated that compared with the control group (gray value of 1.41±0.41), the level of p-STAT5 of Hep G2 cells of the long-term GH group significantly decreased (gray value of 0.76±0.31) (P=0.026).2. Effect of long-term administration of growth hormone on the level of p-STAT5 in mice livers of in vivo study (animal study).2.1 The experiment results showed that compared with the control group (gray value of 1.47±0.45), the level of p-STAT5 of mice livers of the long-term GH group significantly decreased (gray value of 0.42±0.06). (P=0.016).2.2 The experiment results demonstrated that the level of p-STAT5 of mice livers of the long-term+single dose GH group (gray value of 2.05±0.33) was significantly higher than that of the long-term GH group (gray value of 0.17±0.03). (P=0.01)2.3 The experiment results indicated that the level of p-STAT5 of mice livers of the long-term GH group (gray value of 1.29±0.34) was significantly less than the single dose GH group (gray value of 3.48±0.88). (P=0.036)Conclusion1. From the in vitro level, long-term administration of growth hormone inhibit the phosphorylation of STAT5, resulting in inhibiting the JAK-STAT signaling pathway.2. In the healthy mice model, long-term administration of growth hormone inhibits the phosphorylation of STAT5, resulting in inhibiting the JAK-STAT signaling pathway and finally growth hormone insensibility of mice livers.3. Combined with our early experiment results we suggest that, long-term administration of growth hormone may increase the transcription level of SOCS-3 gene, and thus inhibit the phosphorylation of STAT5, inhibiting the JAK-STAT signaling pathway, and decrease the sensitivity to growth hormone stimulation on liver. |
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