DnaJA4 is Involved in Responses to Hyperthermia by Regulating the Expression of F-Actin in HaCaT Cells

DnaJA4 is Involved in Responses to Hyperthermia by Regulating the Expression of F-Actin in HaCaT Cells

Introduction

Actin is a highly conserved multi-functional protein present in essentially all eukaryotic cells, with an approximate mass of 42 kDa. It exists in two forms: globular actin (G-actin), which is a spherical monomer, and fibrous actin (F-actin), which is a polymer of spherical actin that comprises the principal component of the cytoskeleton. These two forms can reversibly convert to each other under certain physiologic conditions, but only F-actin possesses physiologic activity. Actin participates in a number of critical cellular processes, including the establishment and maintenance of cell junctions and cell shape, cell migration, cell signaling, phagocytosis, apoptosis, and proliferation.

Heat shock proteins (HSPs) are a type of acute reaction proteins that are highly conserved with considerable diversity among species. Their expression increases under stressful conditions such as hyperthermia and ultraviolet B radiation. Among these HSPs, DnaJ/HSP40s, with a molecular weight of approximately 40,000 Da, function as co-chaperones of HSP70 to protect damaged cells. As a chaperone, HSP40 can directly or indirectly affect the replication of viruses. Hyperthermia, defined by a 30- to 60-minute exposure to a thermal stimulus of 40°C to 44°C, has been used to treat various diseases, including breast cancer and non-small cell lung cancer. In clinical settings, local hyperthermia at 44°C for 30 minutes has been found to be more effective than conventional therapies for the treatment of plantar warts (verruca vulgaris).

Previous studies have revealed that DnaJA4 expression in HaCaT cells increases following hyperthermia, and it has been confirmed that hyperthermia affects the anti-viral immunity of HaCaT cells. DnaJA4-knockout (KO) combined with hyperthermia enhances this response. Mass spectrometry analysis has shown that DnaJA4 interacts with cytoskeleton proteins, tubulin, and actin in response to hyperthermia. Therefore, the purpose of this study was to examine the effects of DnaJA4 on F-actin in HaCaT cells in response to hyperthermia.

Methods

Cell Lines and Culture

Wild-type (WT) HaCaT cells were purchased from GENE (Shanghai, China). DnaJA4-KO HaCaT cells were constructed using CRISPR/Cas9 technology. The cells were cultured in high glucose Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and maintained in a humidified incubator at 37°C with 5% CO2.

Hyperthermia Treatment

Cells were inoculated in 60 mm petri dishes or 12-well plates and grown to 60% to 70% cell confluence overnight. The petri dishes or 12-well plates were then immersed in a constant temperature water bath at 44°C (±0.1°C) for 30 minutes. After hyperthermia treatment, cells were returned to the incubator at 37°C for recovery and harvested at varying recovery time points of 6, 12, and 24 hours for further analyses. Both WT and DnaJA4-KO HaCaT cells were subjected to identical treatments.

Staining of F-Actin

Special cell coverslips were soaked in 70% ethanol overnight, washed three times with phosphate-buffered saline (PBS), and placed in 12-well plates. Cells were inoculated onto the coverslips and grown to 60% confluence before being subjected to hyperthermia or remaining unheated at 37°C. Cells were then rinsed with PBS, fixed in 4% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes. Staining of F-actin was performed using phalloidin conjugated to Alexa Fluor 488. Cells were stained for 60 minutes at room temperature in the dark. Nuclear DNA was stained using 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were mounted in anti-fluorescent attenuation mounts and examined using confocal laser scanning microscopy.

Flow Cytometry Assay

WT and DnaJA4-KO cells were trypsinized and counted using a cytometer. Four hundred thousand cells were washed with PBS and fixed in Fixation Buffer in the dark for 20 minutes. Cells were resuspended in Intracellular Staining Perm Wash Buffer and centrifuged twice at 350 × g for 10 minutes. Cells were then incubated with primary antibodies consisting of mouse anti-human F-actin monoclonal antibodies for 1 hour and the secondary antibody being Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin M mu chain for 30 minutes. Finally, all cells were tested using a BD LSRFortessa instrument, and a minimum of 10,000 cells were gated.

Protein Preparation and Western Blotting Assay

WT and DnaJA4-KO cells were harvested at varying recovery time points after hyperthermia treatment. The cells were washed three times with PBS and lysed using radioimmunoprecipitation assay lysis buffer with 1 mmol/L of phenylmethanesulfonyl fluoride, cOmplete, EDTA-free protease inhibitor cocktail, and PHOSstop phosphatase inhibitor. Cell lysates were collected and centrifuged at 15,000 × g at 4°C for 15 minutes. The concentration of protein lysates was measured using the BCA protein assay kit. Protein aliquots were loaded with SDS buffer and boiled at 99°C for 10 minutes. The denatured protein samples were then electrophoresed, transferred to a membrane, blocked, and incubated with antibodies. Primary antibodies included mouse anti-human F-actin monoclonal antibodies, rabbit anti-human RhoA monoclonal antibody, rabbit anti-human rho-associated serine/threonine kinase 1 (ROCK1) monoclonal antibody, rabbit anti-human E-cadherin monoclonal antibody, rabbit anti-human β-catenin monoclonal antibody, and rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibodies. Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse IgM mu chain and horseradish peroxidase-conjugated goat anti-rabbit IgG polyclonal antibody.

Results

DnaJA4-Knockout and Hyperthermia Induced Morphologic Changes of HaCaT Cells

In the unheated group, WT cells showed many filopodia, loose intercellular connections, and dispersed growth. However, fewer filopodia, tight intercellular connections, and cell fusion were observed in DnaJA4-KO cells. In response to hyperthermia, WT cells were shrunken and rounded at 6 hours after hyperthermia but essentially resumed their normal state at 24 hours post-hyperthermia. Additionally, filopodia in these hyperthermia-treated WT cells were increased, achieving maximal numbers at 12 hours after heat treatment. Although supple intercellular connections were observed at 24 hours after hyperthermia, no significant changes in actin cytoskeleton within DnaJA4-KO cells were obtained at 6 and 12 hours after heating.

DnaJA4-Knockout Up-Regulated F-Actin Expression in HaCaT Cells

Flow cytometry was performed following F-actin staining to compare F-actin expression levels in WT cells versus DnaJA4-KO cells. DnaJA4-KO cells showed increased degrees of mean fluorescent intensity (6364.33 ± 989.10 vs. 4272.67 ± 918.50, P = 0.014) compared with WT cells, indicating that greater levels of F-actin expression were present in DnaJA4-KO cells.

DnaJA4-Knockout Up-Regulated the Expression of F-Actin and Related Pathway Proteins

Western blotting was used to analyze the expression of F-actin and related pathway proteins following hyperthermia. In WT cells, compared with the unheated group, F-actin expression decreased at 6 hours after hyperthermia and then increased to the initial unheated treatment baseline levels at 12 hours after hyperthermia, remaining slightly lower than baseline levels at 24 hours after hyperthermia (37°C vs. 44°C-6 h or 44°C-12 h or 44°C-24 h: 0.34 ± 0.02 vs. 0.24 ± 0.01, 0.35 ± 0.02, 0.31 ± 0.01, P = 0.000, P = 0.537, P = 0.034). In DnaJA4-KO cells, compared with the unheated group, F-actin expression also decreased at 6 hours after hyperthermia and gradually recovered at 12 hours after hyperthermia but exceeded those of the baseline unheated condition at 24 hours after hyperthermia (37°C vs. 44°C-6 h or 44°C-12 h or 44°C-24 h: 0.44 ± 0.01 vs. 0.30 ± 0.01, 0.38 ± 0.03, 0.51 ± 0.02, P < 0.001, P = 0.007, P = 0.001). In response to hyperthermia, compared with the unheated group, F-actin expression within both WT and DnaJA4-KO cells showed an initial tendency to decrease followed by an increase after hyperthermia. Furthermore, DnaJA4-KO cells showed an overall greater level of F-actin expression compared with WT cells throughout the post-hyperthermia sampling periods.

When evaluating responses of the upstream factors of F-actin, a similar profile was observed in ROCK1 and RhoA expressions. In response to hyperthermia, compared with the unheated group, their expression within both WT and DnaJA4-KO cells all showed an initial tendency to decrease followed by an increase after hyperthermia. In addition, their overall expressions were also significantly greater in DnaJA4-KO versus WT cells.

In contrast, the expression of E-cadherin in both WT and DnaJA4-KO cells was decreased at 24 hours after hyperthermia (WT cells: 37°C vs. 44°C-24 h: 0.32 ± 0.02, 0.28 ± 0.01, P = 0.020; DnaJA4-KO cells: 37°C vs. 44°C-24 h: 0.50 ± 0.02, 0.47 ± 0.01, P = 0.036). However, the overall expression of E-cadherin in DnaJA4-KO cells remained significantly greater than that in WT cells. The expression of β-catenin was not significantly changed in response to hyperthermia.

Discussion

Hyperthermia destroys cell membranes by disrupting their stability and increasing their permeability. It can also inhibit Na+ and Ca2+ pumps, leading to decreases in intracellular K+ and increases in Ca2+ concentrations. Under such conditions, F-actin tends to be depolymerized into G-actin. Thermotherapy can down-regulate integrin levels, resulting in inhibition of integrin-mediated adhesion kinase activity, which further dephosphorylates adhesion plaque components leading to disintegration and disappearance. As actin is in contact with the adhesive plaque, disruption of adhesive plaques tends to depolymerize microfilaments, thus weakening cell adhesion ability and producing rounded and buoyant cell bodies. Simultaneously, expressions of HSPs are increased after hyperthermia, which serves to protect these damaged cells. Specifically, this stress induces rapid phosphorylation of Hsp27 and promotes F-actin polymerization, thus stabilizing the actin cytoskeleton. In general, following hyperthermia, the actin cytoskeleton is initially depolymerized and shortened, followed by reassembly, polymerization, and some degree of extension, which is the result of the unique assembly dynamics of the actin cytoskeleton. These events are consistent with the profiles of F-actin expression as revealed in Western blots as well as cell morphologic changes observed in immunofluorescent assays. Our findings that DnaJA4-KO increased the aggregation of F-actin in HaCaT cells following heating suggest that DnaJA4 might down-regulate the expression of F-actin in response to hyperthermia.

The Rho family of small GTPases is key regulatory molecules linking cell membrane surface receptors with the actin cytoskeleton. Within this family, RhoA, Rac1, and Cdc42 are the best studied. RhoA induces stress fibers and enhances focal adhesions, while Cdc42 and Rac1 contribute to the formation of filopodia and lamellipodia, respectively. In the Rho/ROCK signaling pathway, RhoA can induce ROCK activation. Activation of the downstream LIM domain kinase phosphorylates cofilin, which then inhibits the depolymerization of F-actin or further activates downstream myosin light chain phosphorylation to promote the formation of stress fibers. In this experiment, ROCK1 and RhoA, as the upstream factors of F-actin, their expression is consistent with F-actin. It suggests that DnaJA4 affects the expression of F-actin-related RhoA/ROCK1 pathway proteins.

Our current results support the previous findings of Sun et al., who reported that hyperthermia reduced HaCaT cell proliferation-induced cell senescence and promoted cytokine expressions responsible for anti-viral activity through a nuclear factor kappa-B-dependent pathway. DNAJA4-deficiency enhanced the activation of nuclear factor kappa-B by hyperthermia in HaCaT cells. Yin et al. proposed that an over-expression of CRYAB (a subtype of small HSPs) may significantly increase the heat resistance of H9C2 cardiomyocytes by reducing F-actin aggregation, regulating the cell cycle, and inhibiting caspase-mediated apoptosis. In this study, DnaJA4-KO increased the aggregation of F-actin in HaCaT cells following hyperthermia. Therefore, DnaJA4 may also be involved in hyperthermia response by regulating the expression of F-actin to affect the apoptosis or proliferation of HaCaT cells. We also found that filopodia were significantly reduced in DnaJA4-KO cells, while the virus could be transmitted to the cell body along the filopodia, leading to cell infection. We wondered whether the reduced filopodia caused by DnaJA4 gene deletion might affect the transmission rate of the virus in vivo.

E-cadherin is an essential adhesion molecule on cell surfaces. It participates in intercellular adhesion and signal transduction through the binding of the cadherin/catenin adhesion complex and actin filaments to the cytoskeleton. In this study, although E-cadherin expression was decreased in both WT and DnaJA4-KO cells, their expression was greater in DnaJA4-KO cells. These findings were consistent with results obtained in the immunofluorescent experiment. It has been reported that excessive expression of E-cadherin in epithelial cells inhibits cell migration and proliferation and induces apoptosis, while down-regulation of this adhesion molecule weakens cell connections, stimulates cell regeneration and migration, and facilitates tissue repair, which then contributes to the maintenance of normal functioning within epithelial cells. Based upon these findings, we speculated that DnaJA4 may affect the proliferation or apoptosis of HaCaT cells by regulating E-cadherin expression in response to hyperthermia. This hypothesis will require verification with future experiments.

Conclusion

In summary, DnaJA4 was involved with modulating responses to hyperthermia in HaCaT cells by regulating the expression of F-actin and the related pathway proteins. Future work, including the establishment of a cell model of DnaJA4 over-expression, will be critical for a better understanding of these mechanisms and to address some of the limitations in our current study.

doi.org/10.1097/CM9.0000000000001064

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