The Role Of Chronic Growth Hormone In Hepatic STAT5Signaling

BackgroundGrowth hormone(GH)is an important homone. GH plays an important role in the regulation of postnatal growth, metabolism, cell aging and tumorigenesis.Recombinant human growth hormone(rhGH)is initially used to treat patients with growth hormone deficiency (GHD) as hormone replacement therapy. Now it has been widely used in various diseases, such as fistula of intestine, Cirrhosis and portal hypertension, acute necrotizing pancreatitis, serious infection, dilated cardiomyopathy, respiratory failure, chronic kidney disease, etc. Its side effects have caused more and more attention. The liver is the most important target organ of GH. GH binds to GHR, causing the dimerization of GHRs, which results in the autophosphorylation of tyrosine kinase JAK2. After dimerization, phosphor-STAT5(p-STAT5) translocates to the nucleus, and activates gene transcription. Studies on the regulation of GH-JAK/STAT5signaling pathway have been conduced in various levels, such as the GHR level, and post-receptor level including the regulator suppressor of cytokine signaling(SOCS)family and Src homology2domain containing phosphotyrosine phosphatase2(SHP2).In this study, we explored the effects of acute and chronic GH on hepatic STAT5signaling. We conducted both shorterm and longterm study and in both cell culture model and animal model. For the animal study, BALB/c mice received continuous rhGH stimulation for three weeks or a single dose rhGH for20minutes before sacrifice. They can provide experimental evidences for the possible effects and mechanisms of longterm GH application on the liver.ObjectiveIn the present study, we investigated the effect of acute and chronic GH on the regulation of STAT5signaling pathway both in vivo and vitro, and further explored the mechanism of GH resistance and the possible role of SHP2in this process.MethodsPart one:Cellular culture study1. HepG2cells were cultured in DMEM medium containing10%fetal bovine serum, and then the cells were evenly passaged to four wells in twelve well plates, two of which were experimental group (acute GH group), and the other two of which were control group. When the cells grew to cover approximately90%areas in the plates, do serum starvation for12hours. Twenty minutes before cell lysis, the experimental group was stimulated by GH (GH saving concentration was1.33mg/ml, working concentration was2000ng/ml, diluted in serum free DMEM medium), while control group received DMEM at the same volume. Cells were lysed with RIPA buffer, and total protein was prepared for the test of activation of STAT52. HepG2cells were cultured in DMEM medium containing10%fetal bovine serum, and passaged on twelve well plates. Two wells were for experimental group (chronic GH group); the others for control group. When the cells grew to approximately90%areas, do serum starvation12hours, Six hours prior to cell lysis, chronic GH group was given GH stimulation, while control group was given the same volume of DMEM, twenty minutes before cell lysis, GH stimulation was given to four wells. Then cells were cracked by RIPA, total proteins were extracted by centrifugation. Detect the p-STAT5and STAT5proteins levels by Western blot analysis.3. The cultivated HepG2cells were evenly passaged into two60mm Petri dishes, and they were randomly divided into GH group and control group. The cells were starved for12hours, followed by6-hour stimulation of GH before harvest. Then cells were lysed with CO-IP Lysis buffer, and total proteins were centrifuged at3000RPM. The proteins were co-incubated with CIS antibody and protein A/G beads; the sample was tested with Western blot using anti-GHR antibody, bloted with antibody to CIS4. Statistical Analysis. All data were expressed as the metic mean±standard deviation (M±SD). The difference between two groups was analyzed by independent t-test or Satterthwaite approximate t-test. P<0.05was considered to be significant.Part two:Animal study1. Study designs. A total of32male Balb/c mice aging from6to8weeks were purchased from the animal center of Southern Medical University, and were randomly divided into PBS control group and chronic GH group. Chronic GH group received3-week administration of recombinant human GH at lμg/g body weight, while control group received PBS at the same volume. After3-week administration of GH, fasting weights were measured, and both chronic GH group and PBS control group were randomly divided into acute GH and non-acute GH group. Mice in the acute GH group received intraperitoneal rhGH20minutes before sacrificed, while the non-acute GH group received PBS. Liver tissues were collected under the condition of anesthesia, and were frozen immediately in the liquid nitrogen, and stored in the-80centigrade refrigerator.2. Compare weights of PBS control group and chronic GH group.3. Compare hepatic steady STAT5and p-STAT5expression between PBS control group and chronic GH group. Hepatic lysates of non-acute GH group (PBS+PBS control group, Chronic GH+PBS group) were collected at low temperature, and serum specific for STAT5and p-STAT5were used to detect related protein expression.4. Test the effect of acute GH stimulation on the phosphorylation of STAT5in chronic GH group. Hepatic lysates of chronic GH group (Chronic GH+PBS group, Chronic GH+acute GH group) were collected at low temperature, and serum specific for STAT5and p-STAT5were used to detect related protein expression.5. Compare acute GH stimulated p-STAT5between PBS control group and chronic GH group in the liver. Hepatic lysates of acute GH group (PBS+acute GH group, Chronic GH+acute GH group) were collected at low temperature, and serum specific for STAT5and p-STAT5were used to detect related protein expression.6. Compare hepatic GHR and SHP2expression of PBS control group and chronic GH group under basic condition. Hepatic lysates of non-acute GH group (PBS+PBS control group, Chronic GH+PBS group) were collected at low temperature, and serum specific for GHR and SHP2were used to detect related protein expression.7. Compare the combination of GHR with SHP2between PBS group and chronic GH group under basic condition. Hepatic lysates of non-acute GH group (PBS+PBS control group, Chronic GH+PBS group) were collected as stated in the CO-IP protocol, and were immunoprecipitated with serum specific for SHP2, and the immunoprecipitates were western blotted with serum specific for GHR to test the GHR expression.8. Statistical Analysis. All data were presented as the arithmetic mean±standard deviation(M±SD). Difference between groups were analyzed by two independent t-test or Bonferroni. P<0.05or P<0.017was considered to be significant. ResultsPart one:Cell culture study1. The p-STAT5in acute GH group was significantly higher than that in the control group (0.513±0.052vs0.029±0.008, t=-22.649,P=0.000). On the contrary, the STAT5between the two groups had no significant differences (1.225±0.088vs.1.146±0.102, t=-1.473,P=0.181).2. The p-STAT5in chronic GH group was lower than that in the control group(0.237±0.043vs0.959±0.298, t=5.868,P=0.000). In the contrast, the STAT5between two groups showed no significant difference (0.875±0.086vs0.788±0.079, t=-1.824,P=0.098). GAPDH was used as the internal control.3. CO-IP study showed that GH-treated group had higher binding capacity between the GHR and the CIS, compared with the control group (1.896±0.193vs0.222±0.075, t=14.016,P=0.000).Part two:Animal study1. Chronic GH administration promoted weight gain in mice. The weight gain in chronic GH group was1.278g higher than that in PBS control group mice (3.883±0.962and2.605±1.008, t=-3.667,P=0.001).2. Steady phosphor-STAT5in chronic GH group was lower than that in the PBS control group (0.367±0.124vs1.058±0.091, t=12.272,P=0.000). The total STAT5protein in the two groups showed no significant differences (0.741±0.157vs0.799±0.086, t=0.927,P=0.370).3.In chronic GH group, acute GH treatment could significantly increased the p-STAT5expression, compared with pre-sacrificed PBS administration (0.214±0.027vs0.005±0.001, t=-21.544,P=0.000). The total STAT5protein in two groups showed no significant differences (0.625±0.046vs0.632±0.060, t=-0.251,P=0.805). 4. Acute GH treatment could both stimulate hepatic STAT5in PBS control group and chronic GH group, while its expression was significantly suppressed in chronic GH group (0.418±0.091vs0.677±0.080, t=6.036,P=0.000). The total STAT5protein expression in two groups had no significant differences (0.826±0.093vs0.811±0.073, t=-0.360,P=0.724).5. Under the condition of pre-sacrificed PBS treatment,the total GHR protein showed no significant difference between PBS control group and chronic GH group (1.280±0.297vs1.178±0.368, t=0.611,P=0.551). The expression of total SHP2protein showed no significant differences (1.297±0.287vs1.400±0.272, t=-0.741,P=0.471).6. Under the condition of pre-sacrificed PBS treatment,the binding of GHR with SHP2was higher in chronic GH group, compared with that in the PBS control group (0.537±0.090vs0.282±0.113, t=-4.980,P=0.000)ConclusionChronic GH treatment induced GH/STAT5resistance in HepG2cells possibly due to an increase in the binding of GHR and CIS in cytoplasm. In vivo, chronic GH led to inhibition of p-STAT5expression in mouse liver, and the binding of GHR and SHP2was correspondingly increased. Thus, we concluded that the chronic GH induced GH/STAT5resistance may be due to the increased binding between GHR and SHP2. This is the first hypothesis that the dysfunction of the binding between GHR and SHP2may result in diseases.