DnaJA4 Is Involved In Responses To Hyperthermia By Regulating The Expression Of F-actin In HaCAT Cells

Objectives: Actin is a member of a highly conserved family of multi-functional proteins that form microfilaments,while only F-actin possesses physiological activity.Actin is involved in a number of important cellular processes,with the exception of cell connections and the establishment and maintenance of cell shapes,migration,phagocytosis and cell signaling.It also plays an important role in cell apoptosis and proliferation.Heat shock proteins(HSPs)are a type of acute reaction proteins,which is the focus of current research as a molecular chaperone.Among them,DnaJ/HSP40 functions as co-chaperone of HSP70 to protect damaged cells.In addition,HSP40 acts as a molecular chaperone to affect the replication of a variety of viruses and as an immune adjuvant to regulate the body’s immune response.Within our clinic we have used local hyperthermia(44℃ for 30 minutes)for the treatment of plantar warts(verruca vulgaris).In the team’s previous experiments,it was confirmed that heat and hyperthermia affected the antiviral immunity of HaCaT cells,and DnaJA4 knockout combined with hyperthermia enhanced this response.Meanwhile,mass spectrometry showed that DnaJA4 interacted with the cytoskeleton proteins,tubulin and actin,in response to hyperthermia.Therefore,as an approach to better understand the bases for these responses,the purpose of this report was to examine the effects of DnaJA4 on F-actin in HaCaT cells during hyperthermia and to explored the role of DnaJA4 in hyperthermia from the perspective of cytoskeleton.Methods: 1.Experiment subjects:Wild-type(WT)and DnaJA4 Knock out(KO)HaCaT cells were isolated and divided into two group either unheated group(37℃)or hyperthermia group(44℃ for 30 min)(including 6h,12 h and 24 h after hyperthermia).The cells of the two types were tested under the conditions of 37 °C and assessed at 6,12 and 24 h after hyperthermia exposure.2.Immunofluorescence(IF): F-actin was stained with the specific dye phalloidin to compare the cytoskeleton morphological differences between WT HaCaT cells and DnaJA4 KO HaCaT cells before and after after hyperthermia.3.Flow Cytometry(FCM): The mean fluorescence intensity of F-actin was measured after F-actin antibody staining,and the expression difference of F-actin in WTHaCaT cells and DnaJA4 KO HaCaT cells was compared.4.Western Blot(WB): The expression of F-actin and related pathway proteins RhoA,ROCK1,E-cadherin andβ-catenin were detected,and the expression differences of the above proteins before and after hyperthermia were compared in WT HaCaT cells and DnaJA4 KO HaCaT cells.Results: 1.Immunofluorescence(IF):In the unheated condition,WT HaCaT cells showed fewer filopodia and increased numbers of intercellular connections than that observed in DnaJA4 KO cells.In response to hyperthermia,intercellular connections in both WT cells and DnaJA4 KO cells were reduced,while filopodia in WT cells were increased as compared with that observed in unheated cells.2.Flow Cytometry(FCM): Compared with WT cells,increased degrees of mean fluorescent intensities were observed in DnaJA4 KO cells(P < 0.01),indicating that greater levels of F-actin expression were present in these DnaJA4 KO cells.3.Western Blot(WB):(1)In response to hyperthermia,compared with the unheated group,F-actin expression within both WT cells and DnaJA4 KO cells showed an initial tendency to decrease followed by an increase after hyperthermia.It embodied in: F-actin expression decreased at 6 hours after hyperthermia,and these expression levels increased to that approaching levels in the unheated group at 12 hours after hyperthermia,but at 24 h after hyperthermia,we found that F-actin expression in wild type cells was restored to levels observed in the unheated condition,while DnaJA4 KO cells showed levels that exceeded the unheated condition.Furthermore,DnaJA4 KO cells showed an overall greater level of F-actin expression as compared with that of WT cells throughout the post-hyperthermia sampling periods.This indicated that DnaJA4 knockout increased the expression of F-actin in HaCaT cells during hyperthermia.(2)When evaluating responses of the upstream factors of F-actin,a similar profile was observed in ROCK1 and RhoA expressions as determined at 6,12 and 24 hours after hyperthermia.In addition,their overall expressions were significantly greater in DnaJA4 KO versus WT cells,which were consistent with F-actin.(3)In contrast,the expression of E-cadherin in both WT cells and DnaJA4 KO cells was decreased throughout all time periods following hyperthermia.However,the overall expression of E-cadherin in DnaJA4 KO cells remained significantly greater than that in WT cells,and there was no significant change in β-catenin.Conclusions: Hyperthermia affects the expression of F-actin in HaCaT cells.DnaJA4 knockout increases the expression of F-actin in HaCaT cells during hyperthermia.DnaJA4 regulates the expression of F-actin and related pathway proteins in response to hyperthermia in HaCaT cells.