The Molecular Mechanism Of ACVR1 And Its Downstream AQP5 Gene Network In Primary Focal Hyperhidrosis

Purpose Primary focal hyperhidrosis(PFH)is a state of hypersecretion of local sweat glands in the body,which has a serious impact on patients’ work,study and social contact.This study aims to clarify the influence of overexpression of ACVR1 on the pathogenesis of PFH and to further reveal the mechanism in order to provide new ideas for the treatment of PFH.Materials and Methods1.Verify the clinical relevance between ACVR1 gene and PFH: Thirty cases of axillary sweat gland tissues from PFH patients(experimental group)and control group without axillary sweat(control group)were collected and divided into three parts.Western Blot,immunohistochemistry and fluorescence quantitative RT-PCR were used to detect the expression difference of ACVR1 in axillary sweat glands.Finally,the clinical relevance between ACVR1 and PFH was analyzed.2.The co-expression of ACVR1 and CK7-the biomarker of sweat gland cells from both experimental group and control group were analyzed by immunofluorescent double labelled staining with laser confocal microscopy.Moreover,the expression differences of ACVR1 from experimental group and control group were compared.3.Axillary sweat gland cells from experimental group were separated using enzymatic digestion.And in-vitro models of axillary sweat gland cells from PFH patients were established.4.The models of ACVR1 overexpression and ACVR1 knockdown were established by exogenous gene overexpression and RNAi technology,respectively.Moreover,cell proliferation,cell apoptosis and cell cycle were analyzed by MTT method and flow cytometry.Afterwards,the genetic functional and phenotypic changes induced by ACVR1 overexpression and knockdown were testified by RT-q PCR and Western blot,respectively.5.The interactive proteins with ACVR1 were discovered by CO-IP.6.Differential proteins from experimental group and control group were discovered by proteomic analysis,followed by GO enrichment.7.The Ach amounts in the serum of experimental group and control group were analyzed by ELISA.Results1.The Western blot result indicated the protein expression in sweat gland tissues from the experimental group significantly increased(P<0.01)compared with the control group.Moreover,the results of immunohistochemistry confirmed that ACVR1 was mainly expressed on the cell membrane of sweat gland cells.And the relative grey intensity of ACVR1 also significantly increased compared with the control group(P<0.01).Meanwhile,the results of RT-q PCR were consistent with other experiments,where the ACVR1 m RNA expression in sweat gland tissues from experimental group significantly enhanced(P<0.001).2.The results of laser confocal microscopy suggested that compared with the control group,the expression level of the biomarker of sweat gland cells CK7 in sweat gland cells from experimental group did not display obvious variation.However,the ACVR1 protein expression in the sweat gland cells from experimental group significantly improved(P<0.01),indicating the clinical relevance of ACVR1 and PFH.3.By using western blot and RT-q PCR,we confirmed that we have successfully established the ACVR1 overexpression vector.Moreover,the result of MTT method indicated that ACVR1 overexpression could trigger the proliferation of sweat gland cells.Besides,the results of flow cytometry and the activity of caspase-3 as well as caspase-8 suggested that the cell apoptosis rate of the cells with ACVR1 overexpression was significantly lower than that of the control group.The cell cycle analysis also confirmed that ACVR1 overexpression could induce more cells to transform from G1 state to S state,which could promote cell proliferation.The knock-down result is the opposite.4.By using western blot and RT-q PCR,we found that ACVR1 overexpression could lead to improved expressions of both AQP5(P<0.01)and NKCC1(P<0.01)at the m RNA level and the protein level.Conversely,ACVR1 knockdown could lead to decreased expressions of both AQP5 and NKCC1 at the m RNA level(P<0.01)and the protein level(P<0.05,P<0.01).5.By CO-IP,we discovered some interactive proteins with ACVR1,among which NT5E、RPS26、CAVIN1、PRR14、RPL39、RPL39P5 and RPL10 came as the most relative ones.6.The results of proteomic analysis revealed that differential proteins mainly participated in biofunctional activities including catalytic activity,binding and transformative activity.GO enrichment indicated that proteins associated with the transportation of inorganic ions were significantly enriched.Meanwhile,it was also discovered that proteins related with the calcium signal pathway including GNAQ、PLCB3、CALML5、CALM3、ACVR1、CACNA1C、NKCC1 and AQP5 remarkably differentiated.The result of Parallel reaction monitoring(PRM)also demonstrated these differential proteins,the relevant pathways were associated with hormone regulation.7.The results of ELISA indicated that compared with the control group,the Ach levels in the serum from PFH patients with axillary hyperhidrosis significantly enhanced(P<0.05).Conclusions1.The ACVR1 overexpression was clinically relevant with PPH.Compared with the control group,the ACVR1 expression of sweat gland tissues from the experimental group significantly enhanced at both m RNA and protein levels.2.By successfully established in-vitro ACVR1 overexpression model,we found that ACVR1 overexpression could trigger the proliferation of sweat gland cells,induce more cells to transform from G1 state to S state,could promote cell proliferation and limit cell apoptosis,while knockdown did the opposite.3.The ACVR1 overexpression could lead to improved expression of both AQP5 and NKCC1 at the m RNA level and the protein level.Conversely,ACVR1 knockdown could lead to decreased expressions of both AQP5 and NKCC1 at the m RNA level and the protein level.Overall,it was indicated that ACVR1 plays a vital role in the process of sweat secretion by sweat glands.4.Proteomic analysis showed that the enrichment results of signal pathway network in which the differential proteins were located involved in hormone regulation and was closely related to sympathetic nervous system regulation.5.The Ach levels in the serum from experimental group significantly enhanced compared with those from control group,indicating that the neurotransmitter delivery during PFH could lead to enhanced expression of ACVR1.6.The possible mechanism of PFH can be the fact that when people were subjected to environmental stimulation such as tension and high temperature,the sympathetic excitability of patients with PFH is enhanced,leading to increased release of Ach by sympathetic nerve endings,which results in the upregulation of CACNA1 C by the calcium signal pathway.As a result,the enhanced release of calcium ion activates the expression of ACVR1 and then upregulates the expression of AQP5 and NKCC1,which finally leads to the excessive sweat secretion of sweat glands.The accurate mechanism underlying PFH still needs further verification.