| BackgroundPituitary adenomas(PAs)are benign tumors originating from the anterior pituitary,accounting for about 15% of all primary brain tumors.PAs are characterized by increased tumor volume and hormone secretion disorders.According to secreted hormone types,PAs can be divided into prolactin(PRL)over-secretion prolactinomas,growth hormone(GH)over-secretion somatotropinomas,thyrotropin(TSH)over-secretion thyrotropinomas and adrenocorticotropic hormone(ACTH)over-secretion corticotropinomas.Excessive hormone secretion seriously threatens the health of patients.At present,surgery and drugs are the main methods for the treatment of PAs.However,the 10-year recurrence rate after surgical resection in patients with PAs is still as high as 7-12%.Therefore,it is of great essential to comprehensively understand the potential molecular biological characteristics and mechanisms of hormone over-expression and over-secretion in PAs,so as to develop effective treatment strategies to control hormone hypersecretion of PAs.Neuregulins(Nrgs)belong to the epidermal growth factor-like family,and they are the largest subclass of transmembrane polypeptide growth signaling molecules.Nrgs act as ligands of Erb B tyrosine kinase receptors(Neu,Erb B3 and Erb B4)to regulate biological processes of various different tumors.At least four NRGs members have been identified,including NRG1,NRG2,NRG3 and NRG4,which encode responding transmembrane signal proteins Nrg1,Nrg2,Nrg3 and Nrg4.Accumulated evidence demonstrates that Nrg1β regulates PRL expression in PA cells,while showing no apparent effect on the GH expression.However,the expression of other Nrg subtypes in PAs and their roles in regulating PRL and GH have not been fully clarified.Whether hormone-secreting pituitary tumor cells can express Nrg2,Nrg3,and Nrg4,thereby regulating PRL and GH secretion,still remains to be elucidated.It is thus important to further explore the roles of Nrg2,Nrg3 and Nrg4 in the development and hormone secretion of PAs.Further study to clarify the role of Nrg2,Nrg3 and Nrg4 in the occurrence,development and expression,and secretion of PRL and GH of PAs may provide new treatment strategies for patients with prolactinomas and somatotropinomas.ObjectiveIn this study,we aimed to identify an effective target molecule and explore the regulatory mechanisms of regulating over-expression and-secretion of PRL and GH on the basis of exploring and comparing the roles of Nrg1,Nrg2,Nrg3 and Nrg4 in GH3 cells and RC-4B/C cells.Methods1.Firstly,the expression of NRG1、NRG2、NRG3 and NRG4 in normal human pituitary tissue samples was evaluated using Human Protein Atlas(HPA)database.Public Gene Expression Omnibus(GEO)database was used to identify the expression of NRGs in human PAs and normal pituitary tissue.Reverse Transcription-Polymerase Chain Reaction and Western blot analysis were performed to verify the gene and protein expression of Nrgs family in rat cortex tissue,rat pituitary tissue,rat pituitary adenomas GH3 cells and RC-4B/C cells.The localization and expression of Nrg2,Nrg3 and Nrg4 in normal rat pituitary tissue and rat pituitary adenoma cells were detected by immunofluorescence technique.2.Different experiments were performed to identify the function of Nrgs in PAs.(1)Cell Counting Kit-8(CCK-8)assay was used to detect the effect of exogenous Nrg1β,Nrg2,Nrg3 and Nrg4 on the proliferation in GH3 cells and RC-4B/C cells.(2)Western blot analysis was performed to analyze the effect of exogenous Nrg1β,Nrg2,Nrg3 and Nrg4 on the expression of p-Neu,Neu,p-Erb B3,Erb B3,p-Erb B4,Erb B4,p-Akt,Akt,p-Erk1/2,Erk1/2,PRL and GH in GH3 cells and RC-4B/C cells.Enzyme Linked Immunosorbent Assay(ELISA)analysis was performed to test the effect of exogenous Nrg1β,Nrg2,Nrg3 and Nrg4 on PRL and GH secretion in GH3 cells and RC-4B/C cells.(3)Western blot analysis was performed to investigate the expression of p-Akt,Akt,PRL and GH proteins in GH3 cells and RC-4B/C cells pretreated with MK2206,and then combined with different exogenous Nrgs treatment.Western blot analysis was performed to investigate the expression of p-Erk1/2,Erk1/2,PRL and GH proteins in GH3 cells and RC-4B/C cells pretreated with FR180204,and then combined with different exogenous Nrgs treatment.3.Different experiments were performed to explore the important role of Nrg4 in PAs cells.(1)Gene Expression Profiling Interactive Analysis(GEPIA)database was used to analyze the correlation between NRGs and PRL expression in 283 normal human pituitary tissues samples.The expression of NRG4 and PRL in normal human pituitary tissue arrays were assessed by immunofluorescence staining and interactive correlation was further analyzed.(2)Western blot analysis and Immunofluorescence analysis were performed to investigate the expression of Nrg4,PRL and GH proteins in GH3 cells and RC-4B/C cells treated with small interfering RNA(si RNA)targeting Nrg4.(3)Western blot analysis was used to investigate the expression of Nrg4,MEL-1B-R,p-Erb B4,p-Erk1/2,Erk1/2,PRL and GH in RC-4B/C cells treated with melatonin at different concentrations and the expression of p-Erb B4,p-Erk1/2,Erk1/2,PRL and GH in RC-4B/C cells treated with melatonin and Nrg4.Results1.The expression of NRGs was identified in human and rat normal pituitary tissues and pituitary adenomas.(1)NRG1,NRG2,NRG3 and NRG4 are expressed in 283 human pituitary tissue samples in HPA database.The expression of NRG1 and NRG2 increased in human PAs samples in GEO database.However,we failed to analyze the expression of NRG3 and NRG4 due to their absence in the test gene list of the selected datasets.(2)RT-PCR analysis showed that the expression of Nrg1,Nrg2,Nrg3 and Nrg4 in GH3 cells was higher than that in rat pituitary tissue.The expression of Nrg1 and Nrg3 in RC-4B/C cells was higher than that in rat pituitary tissue.In contrast,the expression of Nrg2 and Nrg4 showed no significant change compared with that in rat pituitary tissue.(3)Western blot analysis showed that Nrg1,Nrg2,Nrg3 and Nrg4 were differentially expressed in normal rat pituitary tissue and PA cells.The Nrg1 antibody identified various bands with molecular weights at about 130,60,45 and 30 k Da in the rat pituitary tissue.Multiple Nrg1 isoforms were identified in both GH3 and RC-4B/C cells,and they were expressed as bands with molecular weights at about 180,130,60,50,45 and 30 k Da.The Nrg1 protein level was obviously higher at apparent molecular weights of 180,60,50,45 and 30 k Da in GH3 cells than that in rat pituitary tissue.The Nrg2 antibody identified two bands with molecular weights at about 60 and 40 k Da in the rat pituitary tissue,GH3 cells and RC-4B/C cells.The Nrg2 protein level was apparently higher at the molecular weight of 40 k Da in GH3 cells than that in rat pituitary tissue and RC-4B/C cells.Nrg3 protein with molecular weight at about 50 k Da was detected in rat pituitary tissue,which was higher than that in GH3 cells and RC-4B/C cells.Nrg3 protein with molecular weights at about 130 and 70 k Da was detected in GH3 cells and RC-4B/C cells,which was higher than that in rat pituitary tissue.The Nrg4 antibody identified a band with molecular weight at about 45 k Da in the rat pituitary tissue,GH3 cells and RC-4B/C cells.In addition,Nrg4 protein with molecular weight at about 70,50,35,25 and 15 k Da was detected in GH3 cells and RC-4B/C cells,which was higher than that in rat pituitary tissue.(4)Immunofluorescence analysis showed that Nrg2 was mainly expressed in ACTH cells,suggesting Nrg2 may regulate the expression of PRL in a paracrine manner.Nrg3 was expressed in GH cells and PRL cells,suggesting Nrg3 may regulate the expression of PRL in autocrine and paracrine manners.Nrg4 was expressed in GH cells,but was weakly expressed in PRL cells,suggesting Nrg4 may regulate the expression of PRL in a paracrine manner.Immunofluorescence analysis showed that Nrg1 and Nrg4 were highly expressed in GH3 cells and RC-4B/C cells.However,the expression of Nrg2 and Nrg3 was weak in GH3 cells and RC-4B/C cells.2.This study explored and compared the role of Nrgs family in cell proliferation,Erb B receptor activation,general signal activation,PRL and GH expression and secretion through different experimental methods.At the same time,according to the intracellular signal activation and the expression of PRL and GH,different signal inhibitors were used to explore their different pathological mechanisms.(1)CCK-8 assay showed that exogenous Nrg1β,Nrg2,Nrg3 and Nrg4 had no effect on proliferation of both GH3 cells and RC-4B/C cells.(2)Different Nrgs diversely promoted the phosphorylated activation of Erb B receptors(Neu,Erb B3 and Erb B4)in GH3 and RC-4B/C cells.Western blot analysis showed that Nrg1βevidently increased phosphorylated Neu level and phosphorylated Erb B4 level in GH3 cells and RC-4B/C cells.Nrg2 obviously increased phosphorylated Erb B3 level and phosphorylated Erb B4 level in GH3 cells.Nrg2 obviously promoted phosphorylated activation of Neu,Erb B3 and Erb B4 in RC-4B/C cells.Nrg3 significantly elevated phosphorylated Erb B4 level in GH3 cells.Nrg3 significantly enhanced phosphorylated activation of Erb B3 and Erb B4 level in RC-4B/C cells.Nrg4 significantly increased the phosphorylation level of Erb B4 in the GH3 cells and those of Erb B3 and Erb B4 in RC-4B/C cells.(3)Nrgs diversely promoted the phosphorylated activation of different Nrgs-related signaling molecules in GH3 and RC-4B/C cells.Western blot analysis showed that Nrg1β stimulated a significant increase in Akt and Erk1/2 phosphorylation in GH3 cells and RC-4B/C cells.Nrg2 significantly promoted an evident increase in Akt and Erk1/2 phosphorylation in GH3 cells.Although Nrg2 stimulated a significant increase in Erk1/2 phosphorylation in RC-4B/C cells,it failed to activate the phosphorylation of Akt in RC-4B/C cells.Nrg3 significantly elevated phosphorylation levels of Akt and Erk1/2 in GH3 cells and that of Erk1/2 in RC-4B/C cells,but it failed to activate Akt phosphorylation in RC-4B/C cells.Nrg4 stimulated a significant increase in Erk1/2 phosphorylation in GH3 cells and RC-4B/C cells,but it failed to activate the phosphorylation of Akt in RC-4B/C cells.(4)Nrgs isoforms differentially affected the expression of PRL and GH in PA cells.Western blot analysis showed that Nrg1β and Nrg4 significantly increased the PRL expression in GH3 cells and RC-4B/C cells.Nrg2 and Nrg3 significantly increased the PRL expression in RC-4B/C cells,but had no influence on that in GH3 cells.The results showed that in GH3 cells,the promoting effect of NRG1β on PRL expression was greater than Nrg4,whereas Nrg2 and Nrg3 had no significant effect on PRL expression in GH3 cells.In RC-4B/C cells,the promotion degree of Nrgs in the expression of PRL is in the following order as Nrg1β> Nrg2>Nrg4>Nrg3.(5)Nrgs isoforms differentially affected the secretion of PRL and GH in PA cells.ELISA analysis showed that Nrg1β,Nrg3 and Nrg4 significantly increased the PRL secretion in GH3 cells and RC-4B/C cells.Nrg2 had no influence on the PRL secretion in GH3 cells and RC-4B/C cells.In GH3 cells and RC-4B/C cells,the promotion degree of Nrgs in the secretion of PRL is in the following order as Nrg4>Nrg3>Nrg1β.(6)All Nrgs had no influence on GH expression and secretion in GH3 cells and RC-4B/C cells.(7)MK2206 pre-treatment significantly reduced p-Akt levels and PRL expression induced by Nrg1β in GH3 cells and RC-4B/C cells.FR180204 pre-treatment significantly reduced p-Erk1/2 levels and PRL expression induced by Nrg1β,Nrg2 and Nrg4 in RC-4B/C cells.However,FR180204 pre-treatment exhibited no detectable effect on PRL expression compared with RC-4B/C cells solely treated with Nrg3.3.Timportant role of Nrg4 was verified in the regulation of PRL in GH3 and RC-4B/C cells.(1)GEPIA database analysis showed that NRG1 and NRG4 were significantly positively correlated with PRL expression in human normal pituitary tissues,while NRG2 and NRG3 were not correlated with PRL expression.The expression of NRG4 was positive correlated with that of PRL in a human pituitary tissue array.(2)Western blot and immunofluorescence analysis showed that inhibition of Nrg4 by si RNA targeting Nrg4 significantly inhibited the protein expression of Nrg4 and PRL.(3)Melatonin promoted the expression of Nrg4,MEL-1B-R,p-Erb B4,p-Erk1/2 and PRL in RC-4B/C cells in the dark.Combined administration of exogenous Nrg4 and melatonin significantly further promoted Erk1/2 phosphorylated activation and PRL expression in RC-4B/C cells compared with those solely treated with Nrg4 or melatonin in the dark.Conclusions1.Nrg1 are highly expressed in the GH3 and RC-4B/C cells.Nrg1β can activate the phosphorylation of Neu and Erb B4,subsequently promoting the phosphorylated activation of Akt and Erk1/2 signaling molecules to enhance PRL expression in GH3 cells and RC-4B/C cells.2.Nrg2 is expressed in ACTH cells,suggesting Nrg2 may regulate the expression of PRL in a paracrine manner in rat pituitary tissue.Nrg2 can activate the phosphorylation of Neu,Erb B3 and Erb B4,subsequently promoting the phosphorylated activation of Erk1/2 signaling molecule to enhance PRL expression in RC-4B/C cells.3.Nrg3 is expressed in GH cells and PRL cells,suggesting Nrg3 may regulate the expression of PRL in autocrine and paracrine manners in rat pituitary tissue.Nrg3 can activate the phosphorylation of Erb B3 and Erb B4,subsequently increasing p-Erk1/2 and PRL expression,but Erk1/2 signaling is not involved in Nrg3 regulation of PRL expression.4.Nrg4 is expressed in GH cells,but expressed weakly in PRL cells,suggesting Nrg4 may regulate the expression of PRL in a paracrine manner in rat pituitary tissue.However,NRG4 was highly expression in human pituitary PRL cells and the expression of Nrg4 and PRL is significantly positively correlated,suggesting Nrg4 may regulate the expression of PRL in an autocrine manner in human pituitary tissue.Nrg4 is highly expressed in GH3 cells and RC-4B/C cells and can activate the phosphorylation of Erb B4,subsequently promoting the phosphorylated activation of Erk1/2 signaling molecule to enhance PRL expression in GH3 cells.Nrg4 can exert a strong promoting effect on PRL expression and secretion.Inhibition of Nrg4 can suppress PRL expression in GH3 cells and RC-4B/C cells.In addition,Nrg4 is involved in the process of PRL expression promotion by melatonin in RC-4B/C cells.Melatonin and Nrg4 can play a synergistic role to jointly mediate the activation of Nrg4-related Erk signaling pathway and promote the expression of PRL in RC-4B/C cells. |
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