HSPA12A promotes c-Myc lactylation-mediated proliferation of tubular epithelial cells to facilitate renal functional recovery from kidney ischemia/reperfusion injury.

Proliferation of renal tubular epithelial cells (TEC) is essential for restoring tubular integrity and thereby to support renal functional recovery from kidney ischemia/reperfusion (KI/R) injury. Activation of transcriptional factor c-Myc promotes TEC proliferation following KI/R; however, the mechanism regarding c-Myc activation in TEC is incompletely known. Heat shock protein A12A (HSPA12A) is an atypic member of HSP70 family. In this study, we found that KI/R decreased HSPA12A expression in mouse kidneys and TEC, while ablation of HSPA12A in mice impaired TEC proliferation and renal functional recovery following KI/R. Gain-of-functional studies demonstrated that HSPA12A promoted TEC proliferation upon hypoxia/reoxygenation (H/R) through directly interacting with c-Myc and enhancing its nuclear localization to upregulate expression of its target genes related to TEC proliferation. Notably, c-Myc was lactylated in TEC after H/R, and this lactylation was enhanced by HSPA12A overexpression. Importantly, inhibition of c-Myc lactylation attenuated the HSPA12A-induced increases of c-Myc nuclear localization, proliferation-related gene expression, and TEC proliferation. Further experiments revealed that HSPA12A promoted c-Myc lactylation via increasing the glycolysis-derived lactate generation in a Hif1α-dependent manner. The results unraveled a role of HSPA12A in promoting TEC proliferation and facilitating renal recovery following KI/R, and this role of HSPA12A was achieved through increasing lactylation-mediated c-Myc activation. Therefore, targeting HSPA12A in TEC might be a viable strategy to promote renal functional recovery from KI/R injury in patients.

Roles of heat shock protein A12A in the development of diabetic cardiomyopathy.

Long-term hyperglycemia can lead to diabetic cardiomyopathy (DCM), a main lethal complication of diabetes. However, the mechanisms underlying DCM development have not been fully elucidated. Heat shock protein A12A (HSPA12A) is the atypic member of the Heat shock 70kDa protein family. In the present study, we found that the expression of HSPA12A was upregulated in the hearts of mice with streptozotocin-induced diabetes, while ablation of HSPA12A improved cardiac systolic and diastolic dysfunction and increased cumulative survival of diabetic mice. An increased expression of HSPA12A was also found in H9c2 cardiac cells following treatment with high glucose (HG), while overexpression of HSPA12A-enhanced the HG-induced cardiac cell death, as reflected by higher levels of propidium iodide cells, lactate dehydrogenase leakage, and caspase 3 cleavage. Moreover, the HG-induced increase of oxidative stress, as indicated by dihydroethidium staining, was exaggerated by HSPA12A overexpression. Further studies demonstrated that the HG-induced increases of protein kinase B and forkhead box transcription factors 1 phosphorylation were diminished by HSPA12A overexpression, while pharmacologically inhibition of protein kinase B further enhanced the HG-induced lactate dehydrogenase leakage in HSPA12A overexpressed cardiac cells. Together, the results suggest that hyperglycemia upregulated HSPA12A expression in cardiac cells, by which induced cell death to promote DCM development. Targeting HSPA12A may serve as a potential approach for DCM management.

Effect of ultrasound-guided thoracic paravertebral block on perioperative analgesia in elderly patients undergoing video-assisted thoracic lobectomy in China: An interventional clinical randomized controlled trial.

BACKGROUND: The aim of this study was to investigate the analgesic effect and safety of ultrasound-guided thoracic paravertebral block (UG-TPVB) in Chinese elderly patients undergoing video-assisted thoracic lobectomy (VATL) and to study the influence of aging factors on these effects. METHODS: This study was a single-center, single-blind, prospective, randomized, controlled trial. A total of 300 patients scheduled for VATL were recruited and randomly divided into the UG-TPVB group (T group) and conventional anesthesia group (C group) according to the recruitment order, and subgroups were set up according to whether the age was ≥65 years old or not. The postoperative 12, 24, and 48 h static/dynamic visual analog scale (VAS) scores, intraoperative fentanyl consumption, postoperative extubation time, post-anesthesia care unit (PACU) stay time, hospitalization days, postoperative complications, and other indicators were compared between the two groups. RESULTS: The postoperative 12, 24, and 48 h static/dynamic VAS scores of the T group were significantly lower than those of the C group. The intraoperative fentanyl consumption, postoperative extubation time, PACU stay time, and postoperative hospitalization days were significantly lower than those of the C group. The incidence of postoperative 48 h urinary retention in the T group was significantly lower than that in the C group. These advantages showed no significant difference or slight difference between elderly patients and nonelderly patients, indicating that UG-TPVB did not influence the analgesic effect and safety of VATL patients by age or age difference. CONCLUSION: UG-TPVB is an effective and safe perioperative analgesia method for elderly VATL patients. Its application improves the quality of life and prognosis of elderly VATL patients.

Hepatocyte HSPA12A inhibits macrophage chemotaxis and activation to attenuate liver ischemia/reperfusion injury via suppressing glycolysis-mediated HMGB1 lactylation and secretion of hepatocytes.

Rationale: Liver ischemia-reperfusion (LI/R) injury is characterized by two interconnected phases: local ischemia that causes hepatic cell damage to release damage-associated molecular pattern (DAMPs), and DAMPs that recruit immune cells to elicit inflammatory cascade for further injury of hepatocytes. High-mobility group box 1 (HMGB1) is a representative DAMP. Studies in macrophages demonstrated that HMGB1 is secreted after lactylation during sepsis. However, whether lactylation mediates HMGB1 secretion from hepatocytes after LI/R is known. Heat shock protein A12A (HSPA12A) is an atypical member of HSP70 family. Methods: Gene expression was examined by microarray analysis and immunoblotting. The hepatic injury was analyzed using released ALT and AST activities assays. Hepatic macrophage chemotaxis was evaluated by Transwell chemotaxis assays. Inflammatory mediators were evaluated by immunoblotting. HMGB1 secretion was examined in exosomes or serum. HMGB1 lactylation was determined using immunoprecipitation and immunoblotting. Results: Here, we report that LI/R decreased HSPA12A expression in hepatocytes, while hepatocyte-specific HSPA12A overexpression attenuated LI/R-induced hepatic dysfunction and mortality of mice. We also noticed that hepatocyte HSPA12A overexpression suppressed macrophage chemotaxis to LI/R-exposed livers in vivo and to hypoxia/reoxygenation (H/R)-exposed hepatocytes in vitro. The LI/R-increased serum HMGB1 levels of mice and the H/R-increased HMGB1 lactylation and secretion levels of hepatocytes were also inhibited by hepatocyte HSPA12A overexpression. By contrast, HSPA12A knockout in hepatocytes promoted not only H/R-induced HMGB1 lactylation and secretion of hepatocytes but also the effects of H/R-hepatocytes on macrophage chemotaxis and inflammatory activation, while all these deleterious effects of HSPA12A knockout were reversed following hepatocyte HMGB1 knockdown. Further molecular analyses showed that HSPA12A overexpression reduced glycolysis-generated lactate, thus decreasing HMGB1 lactylation and secretion from hepatocytes, thereby inhibiting not only macrophage chemotaxis but also the subsequent inflammatory cascade, which ultimately protecting against LI/R injury. Conclusion: Taken together, these findings suggest that hepatocyte HSPA12A is a novel regulator that protects livers from LI/R injury by suppressing glycolysis-mediated HMGB1 lactylation and secretion from hepatocytes to inhibit macrophage chemotaxis and inflammatory activation. Therefore, targeting hepatocyte HSPA12A may have therapeutic potential in the management of LI/R injury in patients.

HSPA12A Stimulates p38/ERK-AP-1 Signaling to Promote Angiogenesis and Is Required for Functional Recovery Postmyocardial Infarction.

Angiogenesis plays a critical role in wound healing postmyocardial infarction (MI). However, there is still a lack of ideal angiogenic therapeutics for rescuing ischemic hearts clinically, suggesting that a more understanding regarding angiogenesis regulation is urgently needed. Heat shock protein A12A (HSPA12A) is an atypical member of the HSP70 family. Here, we demonstrated that HSPA12A was upregulated during endothelial tube formation, a characteristic of in vitro angiogenesis. Intriguingly, overexpression of HSPA12A promoted in vitro angiogenic characteristics including proliferation, migration, and tube formation of endothelial cells. By contrast, deficiency of HSPA12A impaired myocardial angiogenesis and worsened cardiac dysfunction post-MI in mice. The expression of genes related to angiogenesis (VEGF, VEGFR2, and Ang-1) was decreased by HSPA12A deficiency in MI hearts of mice, whereas their expression was increased by HSPA12A overexpression in endothelial cells. HSPA12A overexpression in endothelial cells increased phosphorylation levels and nuclear localization of AP-1, a transcription factor dominating angiogenic gene expression. Also, HSPA12A increased p38 and ERK phosphorylation levels, whereas inhibition of p38 or ERKs diminished the HSPA12A-promoted AP-1 phosphorylation and nuclear localization, as well as VEGF and VEGFR2 expression in endothelial cells. Notably, inhibition of either p38 or ERKs diminished the HSPA12A-promoted in vitro angiogenesis characteristics. The findings identified HSPA12A as a novel angiogenesis activator, and HSPA12A might represent a viable strategy for the management of myocardial healing in patients with ischemic heart diseases.

Heat shock protein A12A activates migration of hepatocellular carcinoma cells in a monocarboxylate transporter 4-dependent manner.

Metastasis is responsible for most of the hepatocellular carcinoma (HCC)-associated death. However, its underlying mechanism has yet to be fully elucidated. Glycolysis-derived lactate has been shown to be a powerful regulator of cancer metastasis. Heat shock protein A12A (HSPA12A) encodes a novel member of HSP70 family. We have recently demonstrated that heat shock protein A12A (HSPA12A) inhibited renal cancer cell migration by suppressing lactate output and glycolytic activity, which were mediated by unstabilizing CD147 and promoting its degradation. By striking contrast, here we demonstrated that HSPA12A promoted migration of human HCC cells. Extracellular acidification, lactate export, and glycolytic activity in HCC cells were also promoted following HSPA12A overexpression. Further analysis revealed that HSPA12A interacted with MCT4 and increased its membrane localization, thereby promoting export of lactate generated from glycolysis; this led, ultimately, to HCC cell migration. Our results revealed the opposite effect of HSPA12A on migration of renal cancer cells and that of HCC cells. Of note, in contrast to the inhibitory effect on CD147 expression in renal cancer cells, we found that HSPA12A increased CD147 expression in HCC cells, indicating that the expression of CD147 might exist heterogeneity in different cancer cell types. Taken together, we identified HSPA12A as an activator of HCC migration, a role opposite to that of renal cancer cells. Inhibiting HSPA12A might be a potential therapeutic intervention for HCC metastasis.

HSPA12A improves endothelial integrity to attenuate lung injury during endotoxemia through activating ERKs and Akt-dependent signaling.

Acute lung injury (ALI) is a critical manifestation of sepsis/septic shock. Disruption of endothelial barrier function is critical for ALI pathogenesis; however, the regulation of endothelial barrier integrity remains largely unclear. Heat shock protein A12A (HSPA12A) is an atypical member of HSP70 family. We have recently demonstrated that hepatocyte HSPA12A attenuated the bacteria endotoxin (lipopolysaccharide, LPS)-induced liver injury. However, the role of HSPA12A in endothelial barrier function and ALI is unknown. Here in this study, HSPA12A showed upregulation in lungs of mice during bacteria endotoxin (lipopolysaccharide, LPS)-induced lung injury in vivo and in primary human umbilical vein endothelial cells (HUVECs) during LPS-induced barrier disruption in vitro. Knockout of HSPA12A in mice exacerbated LPS-induced ALI. Intriguingly, overexpression of HSPA12A in HUVECs attenuated the LPS-induced endothelial hyperpermeability. In line with this, HSPA12A overexpression increased VE-cadherin and decreased VEGF expression following LPS treatment in HUVECs. Also, knockout of HSPA12A enhanced the LPS-evoked pulmonary endothelial cell apoptosis in mice whereas overexpression of HSPA12A inhibited the LPS-induced death of HUVECs. The levels of ERKs and Akt phosphorylation in HUVECs were promoted by HSPA12A overexpression when cells exposed to LPS. Importantly, inhibition of either ERKs or Akt diminished the HSPA12A-induced protection from LPS-induced endothelial hyperpermeability and death. Taken together, these findings indicated that HSPA12A is a novel regulator of endothelial barrier function through both ERKs and Akt-mediated signaling. HSPA12A might represent a viable strategy for the pulmonary protection against endotoxemia challenge.

Safety application of muscle relaxants and the traditional low-frequency ventilation during the flexible or rigid bronchoscopy in patients with central airway obstruction: a retrospective observational study.

BACKGROUND: Bronchoscopy treatments of central airway obstruction (CAO) under general anesthesia are high-risky procedures, and posing a giant challenge to the anesthesiologists. We summarized and analyzed our clinical experience in patients with CAO undergoing flexible or rigid bronchoscopy, to estimate the safety of skeletal muscle relaxants application and the traditional Low-frequency ventilation. METHODS: Clinical data of 375 patients with CAO who underwent urgent endoscopic treatments in general anesthesia from January 2016 to October 2019 were retrospectively reviewed. The use ratio of skeletal muscle relaxants, dose of skeletal muscle relaxants used, the incidence of perioperative adverse events, adequacy of ventilation and gas exchange, post-operative recovery between rigid bronchoscopy and flexible bronchoscopy therapy, and risk factors for postoperative ICU admission were evaluated. RESULTS: Of the 375 patients with CAO, 204 patients were treated with flexible bronchoscopy and 171 patients were treated with rigid bronchoscopy. Muscle relaxants were used in 362 of 375 patients (including 313 cisatracurium, 45 rocuronium, 4 atracurium, and 13 unrecorded). The usage rate of muscle relaxants (96.5% in total) was very high in patients with CAO who underwent either flexible bronchoscopy (96.6%) or rigid bronchoscopy (96.5%) therapy. The dosage of skeletal muscle relaxants (Cisatracium) used was higher in rigid bronchoscopy compared with flexible bronchoscopy therapy (10.8 ± 3.8 VS 11.6 ± 3.6 mg, respectively, p < 0.05). No patient suffered the failure of ventilation, bronchospasm and intraoperative cough either in flexible or rigid bronchoscopy therapy. Hypoxemia was occurred in 13 patients (8 in flexible, 5 in rigid bronchoscopy) during the procedure, and reintubation after extubation happened in 2 patients with flexible bronchoscopy. Sufficient ventilation was successfully established using the traditional Low-frequency ventilation with no significant carbon dioxide accumulation and hypoxemia occurred both in flexible and rigid bronchoscopy group (p > 0.05). Three patients (1 in flexible and 2 in rigid) died, during the post-operative recovery, and the higher grade of American Society of Anesthesiologists (ASA) and obvious dyspnea or orthopnea were the independent risk factors for postoperative ICU admission. CONCLUSION: The muscle relaxants and low-frequency traditional ventilation can be safely used both in flexible and rigid bronchoscopy treatments in patients with CAO. These results may provide strong clinical evidence for optimizing the anesthesia management of bronchoscopy for these patients.

Comparison of the effective orifice area of prosthetic mitral valves using two-dimensional versus three-dimensional transesophageal echocardiography.

OBJECTIVE: This study compared the continuity equation-based effective orifice area (EOA) of prosthetic mitral valves between two-dimensional (2D) and 3D transesophageal echocardiography (TEE). METHODS: Thirty-four patients without major aortic valve abnormalities underwent mitral valve replacement surgery. The EOAs of prosthetic mitral valves were calculated using the continuity equation with 2D and 3D TEE. For 18/34 patients using a biological valve prosthesis, the EOA of the prosthesis was obtained from commercial records. RESULTS: The EOA of prosthetic mitral valves significantly varied between the 2D and 3D methods (2.22 ± 0.71 vs 2.35 ± 0.70 cm2, n = 34). The area of the diameter of the left ventricular outflow tract as determined by the 3D method was significantly higher than that by the 2D method (mean difference: -0.14 ± 0.20 cm2), with 95% coherence boundaries of -0.53 and 0.25 cm2. The regression equation for the EOA by 3D and 2D TEE was y = 0.27 + 0.94x, with a good correlation. CONCLUSIONS: The EOA of prosthetic mitral valves is underestimated using the 2D TEE method compared with the 3D TEE method. The 3D-TEE method has the advantage of higher precision over the 2D TEE method, and it may be helpful for better assessment of prosthetic mitral valves intraoperatively.

Penehyclidine mitigates postoperative nausea and vomiting and intraoperative oculocardiac reflex in patients undergoing strabismus surgery: a prospective, randomized, double-blind comparison.

BACKGROUND: Postoperative nausea and vomiting (PONV) is one of the most frequent complications following strabismus surgery. Penehyclidine, an anticholinergic agent, is widely used as premedication. This study investigated the effect of preoperative penehyclidine on PONV in patients undergoing strabismus surgery. METHODS: In this prospective, randomized, double-blind study, patients scheduled for strabismus surgery under general anesthesia were randomly assigned to either penehyclidine (n = 114) or normal saline (n = 104) group. Penehyclidine was administrated immediately after anesthesia induction, and normal saline was substituted as control. PONV was investigated from 0 to 48 h after surgery. Intraoperative oculocardiac reflex (OCR) was also recorded. RESULTS: Compared with normal saline, penehyclidine significantly reduced PONV incidence (30.7% vs. 54.8%, P < 0.01) and mitigated PONV severity as indicated by severity scoring (P < 0.01). Compared with normal saline, penehyclidine also significantly reduced OCR incidence (57.9% vs. 77.9%, P < 0.01) and mitigated OCR severity, as indicated by the requirement for atropine rescue (77.3% vs. 90.1%, P < 0.05) and the maximum decrease of heart rate during OCR (23.1 ± 9.4 bpm vs. 27.3 ± 12.4 bpm, P < 0.05). The recovery course did not differ between groups. CONCLUSIONS: Penehyclidine administrated after anesthesia induction significantly reduced the incidence of PONV and alleviated intraoperative OCR in patients undergoing strabismus surgery. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT04054479 ). Retrospectively registered August 13, 2019.

Glycogen Synthase Kinase 3ß Promotes Postoperative Cognitive Dysfunction by Inducing the M1 Polarization and Migration of Microglia.

Postoperative cognitive dysfunction (POCD) is a common postoperative central nervous system complication, especially in the elderly. It has been consistently reported that the pathological process of this clinical syndrome is related to neuroinflammation and microglial proliferation. Glycogen synthase kinase 3ß (GSK-3ß) is a widely expressed kinase with distinct functions in different types of cells. The role of GSK-3ß in regulating innate immune activation has been well documented, but as far as we know, its role in POCD has not been fully elucidated. Lithium chloride (LiCl) is a widely used inhibitor of GSK-3ß, and it is also the main drug for the treatment of bipolar disorder. Prophylactic administration of lithium chloride (2 mM/kg) can inhibit the expression of proinflammatory mediators in the hippocampus, reduce the hippocampal expression of NF-κB, and increase both the downregulation of M1 microglial-related genes (inducible nitric oxide synthase and CD86) and upregulation of M2 microglial-related genes (IL-10 and CD206), to alleviate the cognitive impairment caused by orthopedic surgery. In vitro, LiCl reversed LPS-induced production of proinflammatory mediators and M1 polarization of microglia. To sum up these results, GSK-3ß is a key contributor to POCD and a potential target of neuroprotective strategies.

[Effects of different intraoperative lithotomy positions on the absorption of irrigation fluid during bipolar plasmakinetic resection of the prostate].

OBJECTIVE: To investigate the effect of intraoperative lithotomy position (LP) with a head-down tilt (HDT) on the absorption of intraoperative irrigation fluid in patients undergoing bipolar plasmakinetic resection of the prostate (PKRP). METHODS: Eighty BPH patients underwent PKRP, 40 in a conventional 0-degree position (0° LP) and the other 40 in a －10-degree HDT position (－10° LP), with 0.9% saline containing 1% ethanol as intraoperative irrigation fluid. We determined the alcohol concentration in the exhaled breath of the patients with a digital alcohol detector at the start of the operation and every 10 minutes afterwards. Meanwhile we recorded the operation time, the volume of intraoperative intravenous crystalloid infusion and the weight of the resected prostatic tissue, monitored the mean arterial pressure (MAP) and heart rate (HR) at 5 minutes before surgery, 30 minutes after the start of surgery and the end of surgery, and measured the concentrations of Na+, K+, Cl－ and Ca2+ with an arterial blood gas analyzer at 5 minutes before surgery and 1 hour after the start of surgery. RESULTS: There were no statistically significant differences in age, height, body weight and prostate volume, or in intraoperative MAP and HR between the 0° LP and －10° LP groups. Compared with the baseline, at 1 hour after the start of PKRP, the patients in the 0° LP group showed significantly decreased concentrations of K+ (ï¼»3.64 ± 0.29ï¼½ vs ï¼»3.49 ± 0.22ï¼½ mmol/L, P = 0.002) and Ca2+ (ï¼»1.16 ± 0.03ï¼½ vs ï¼»1.13 ± 0.04ï¼½ mmol/L, P = 0.001), increased concentration of Cl－ (ï¼»106.9 ± 2.2ï¼½ vs ï¼»108.7 ± 2.3ï¼½ mmol/L, P = 0.006), but no significant difference in the concentration of Na+ (ï¼»139.7 ± 1.5ï¼½ vs ï¼»139.4 ± 1.6ï¼½ mmol/L, P = 0.231), while those in the －10° LP group exhibited remarkably decreased concentration of Ca2+ (ï¼»1.14 ± 0.04ï¼½ vs ï¼»1.13 ± 0.04ï¼½ mmol/L, P = 0.016) but no statistically significant differences in the concentrations of Na+ (ï¼»140.3 ± 1.8ï¼½ vs ï¼»140.0 ± 2.0ï¼½ mmol/L, P = 0.156), K+ (ï¼»3.49 ± 0.36ï¼½ vs ï¼»3.47 ± 0.34ï¼½ mmol/L, P = 0.506) and Cl－ (ï¼»108.2 ± 2.6ï¼½ vs ï¼»109.1 ± 2.5ï¼½ mmol/L, P = 0.071). Over 1 500 ml of intraoperative irrigation fluid absorption was observed in 6 cases (15%) in the 0° LP group as compared with 4 cases (10%) in the －10°LP group, with no significant difference between the two groups. CONCLUSIONS: Lithotomy position with a 10-degree head-down tilt can reduce PKRP-induced decrease in the concentration of K+ and increase in that of Cl－ without affecting the levels of the other electrolytes.

A new self-designed "tongue root holder" device to aid fiberoptic intubation.

OBJECTIVE: In this study, we aimed to assess the feasibility of fiberoptic intubation (FOI), using a new, self-designed, "tongue root holder" device, in combination with the jaw thrust maneuver. METHODS: Three hundred patients undergoing elective surgery requiring orotracheal intubation were enrolled. Patients presented at least one or more risk factors for difficult airway. The patients were randomly allocated at a 1:1 ratio to one of two groups: group L, FOI with tongue root holder, or group C, standard FOI. Orotracheal FOI was performed after commencement of anesthesia. The jaw thrust maneuver was applied in both groups to facilitate advancement of the fiberoptic bronchoscope. The primary endpoint was the feasibility of FOI. The secondary endpoints were number of attempts, time to intubation, and airway clearance at the soft palate and epiglottis levels. RESULTS: The FOI was achieved in all 150 patients in group L, significantly higher than that in group C (100% vs 95.3%; P = 0.015). Less attempts of intubation were made in group L (P = 0.039). Mean time to successful intubation on the first attempt was shorter in group L (P < 0.001). The mean times to view the vocal cord and carina were also shorter in group L (P = 0.011 and P < 0.001, respectively). Airway clearance was better in group L at both the soft palate and the glottis levels (P = 0.010 and P = 0.038, respectively). CONCLUSIONS: This study shows that FOI is feasible with the newly introduced, self-designed, "tongue root holder" device, when combined with the jaw thrust maneuver in patients with risk factors for difficult airway. The device also provides better airway clearance, less intubation attempts, and shorter time to intubation at first attempt. CLINICAL RELEVANCE: Fiberoptic bronchoscope has been the gold standard for routine management of difficult airway. A technique to open the airway is introduced to reduce the incidence rate of upper airway obstruction.

HSPA12A unstabilizes CD147 to inhibit lactate export and migration in human renal cell carcinoma.

Background: Metastasis accounts for 90% of cancer-associated mortality in patients with renal cell carcinoma (RCC). However, the clinical management of RCC metastasis is challenging. Lactate export is known to play an important role in cancer cell migration. This study investigated the role of heat shock protein A12A (HSPA12A) in RCC migration. Methods: HSPA12A expression was examined in 82 pairs of matched RCC tumors and corresponding normal kidney tissues from patients by immunoblotting and immunofluorescence analyses. The proliferation of RCC cells was analyzed using MTT and EdU incorporation assays. The migration of RCC cells was evaluated by wound healing and Transwell migration assays. Extracellular acidification was examined using Seahorse technology. Protein stability was determined following treatment with protein synthesis inhibitor cycloheximide and proteasome inhibitor MG132. Mass spectrometry, immunoprecipitation, and immunoblotting were employed to examine protein-protein interactions. Results: RCC tumors from patients showed downregulation of HSPA12A, which was associated with advanced tumor node metastasis stage. Intriguingly, overexpression of HSPA12A in RCC cells inhibited migration, whereas HSPA12A knockdown had the opposite effect. Lactate export, glycolysis rate, and CD147 protein abundance were also inhibited by HSPA12A overexpression but promoted by HSPA12A knockdown. An interaction of HSPA12A with HRD1 ubiquitin E3 ligase was detected in RCC cells. Further studies demonstrated that CD147 ubiquitination and proteasomal degradation were promoted by HSPA12A overexpression whereas inhibited by HSPA12A knockdown. Notably, the HSPA12A overexpression-induced inhibition of lactate export and migration were abolished by CD147 overexpression. Conclusion: Human RCC shows downregulation of HSPA12A. Overexpression of HSPA12A in RCC cells unstabilizes CD147 through increasing its ubiquitin-proteasome degradation, thereby inhibits lactate export and glycolysis, and ultimately suppresses RCC cell migration. Our results demonstrate that overexpression of HSPA12A might represent a viable strategy for managing RCC metastasis.

Heat-Shock protein A12A is a novel PCNA-binding protein and promotes hepatocellular carcinoma growth.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. Proliferating cell nuclear antigen (PCNA) plays a pivotal role in cancer development and progression. However, the long-term dismal prognosis of HCC mandates more investigation to identify novel regulators in HCC pathogenesis. Heat-shock protein A12A (HSPA12A) encodes a novel member of the HSP70 family. Here, we report that HCC cells showed increased HSPA12A expression, and overexpression of HSPA12A promoted HCC growth and angiogenesis in mice. Gain- and loss-of-functional studies demonstrated that the proliferation of HCC HepG2 cells, as well as ß-catenin expression and nuclear translocation, was promoted by HSPA12A overexpression, but in turn suppressed by HSPA12A knockdown. HSPA12A did not impact PCNA expression; however, mass spectrometry and co-immunoprecipitation immunoblotting analysis revealed that HSPA12A directly binds to PCNA and promotes its trimerization, which is an essential functional conformation of PCNA for carcinogenesis. Importantly, PCNA inhibition by PCNA-I1 reversed the HSPA12A-mediated HepG2 cell differentiation. These findings indicate that HSPA12A is a novel regulator of HCC cell proliferation and tumor growth through binding to PCNA for its trimerization. HSPA12A inhibition might represent a viable strategy for the management of HCC in humans.

Alpha-lipoic acid inhibits proliferation and migration of human vascular endothelial cells through downregulating HSPA12B/VEGF signaling axis.

Endothelial cells play essential roles in angiogenesis. Heat shock protein A12B (HSPA12B), a novel member of the multigene Hsp70 family, expresses specifically in endothelial cells. Alpha-lipoic acid (LA) has been used for the treatment of human diabetic complications for more than 20 years. However, little is known whether LA impacts endothelial proliferation and migration. To address these questions, primary human umbilical vein endothelial cells (HUVECs) were isolated and treated with LA. We found that LA reduced viable HUVECs but not caused LDH leakage and nuclear condensation, suggesting an inhibitory effect of LA on HUVEC proliferation. We also noticed that LA impeded wound closure of HUVEC monolayers. The expressions of C-Myc, VEGF, and eNOS and phosphorylation of focal adhesion kinase were reduced by LA. Moreover, LA decreased the expression of heat shock protein A12B (HSPA12B). Notably, overexpression of HSPA12B in endothelial cells prevented the LA-induced loss of VEGF. More importantly, HSPA12B overexpression attenuated the LA-induced inhibition of endothelial proliferation and migration. Collectively, the results demonstrated that LA inhibited proliferative and migratory abilities in human vascular endothelial cells through the downregulation of the HSPA12B/VEGF signaling axis. The data suggest that besides the treatment in diabetic complications, LA might represent a viable therapeutic potential for human diseases that involve high angiogenic activities such as cancers.

Alpha-lipoic acid inhibits lung cancer growth via mTOR-mediated autophagy inhibition.

Lung cancer is the leading cause of cancer-related death, and there remains a need for novel therapies for this malignancy. Here, we examined the effects of alpha-lipoic acid (LA), a drug used for treating human diabetic complications, on lung cancer growth. We report that LA limited lung cancer growth in xenograft mice and reduced lung cancer A549 cell viability. We observed autophagy activation in human lung cancers, and report that LA inactivated autophagy in A549 cells. In addition, LA activated mammalian target of rapamycin (mTOR)/p70S6K signaling. Inhibition of mTOR with rapamycin reversed LA-induced inactivation of autophagy and abolished LA-induced suppression of A549 cell viability. Altogether, the data suggest that LA exerts an anti-lung cancer effect through mTOR-mediated inhibition of autophagy, and thus LA may have therapeutic potential for lung cancer management.

Deficiency of heat shock protein A12A promotes browning of white adipose tissues in mice.

Browning of white adipose tissues (WAT) is critical for a variety of physiological and pathophysiological events. Given the limited understanding in molecular control of WAT browning, further research is needed. Heat shock protein A12A (HSPA12A) is a new member of multigene Hsp70 family. This study investigated the effect of HSPA12A on the browning of WAT. WAT Browning in mice was induced by cold exposure for 5 days. We observed that nuclear HSPA12A content was increased in WAT after cold exposure, while deficiency of HSPA12A (Hspa12a-/-) promoted the cold-induced browning of WAT in mice compared to wild type (WT) littermates. Accordingly, Hspa12a-/- mice showed attenuation of body temperature drop and increase of thermogenic gene expression compared to WT mice after cold exposure. However, in vitro experiments demonstrated that HSPA12A deficiency in primary white adipocytes did not affect their browning and thermogenic gene expression. Further loss- and gain-of-HSPA12A functional studies revealed that HSPA12A deficiency promoted whereas HSPA12A overexpression impeded M2 macrophage polarization. Importantly, the conditioned medium (CM) from Hspa12a-/- bone marrow-derived macrophages (BMDMs) enhanced the browning of primary white adipocytes when compared to the CM from WT BMDMs. The data identified macrophage HSPA12A as a novel regulator of WAT browning through a paracrine mechanism. Targeting HSPA12A might provide meaningful advances for the management of browning-associated physiological events such as hypothermia adaptation and pathophysiological disorders such as obesity and cancer-related cachexia.

HSPA12A Is a Novel Player in Nonalcoholic Steatohepatitis via Promoting Nuclear PKM2-Mediated M1 Macrophage Polarization.

Nonalcoholic steatohepatitis (NASH) is the most prevalent cause of chronic liver disease worldwide. Macrophage-mediated inflammation plays a critical role in NASH pathogenesis; however, optimum therapies for macrophage activation and NASH remain elusive. HSPA12A encodes a novel member of the HSP70 family. Here, we report that NASH patients showed increased hepatic HSPA12A expression and serum HSPA12A contents. Intriguingly, knockout of HSPA12A (Hspa12a-/- ) in mice attenuated high-fat diet (HFD)-induced hepatic steatosis and injury. HFD-induced macrophage polarization toward an M1 phenotype and inflammatory responses in the liver of Hspa12a-/- mice were also attenuated. Loss- and gain-of-function studies revealed that the de novo lipogenesis in hepatocytes was regulated by the paracrine effects of macrophage HSPA12A rather than by hepatocyte HSPA12A. In-depth molecular analysis revealed that HSPA12A interacted with the M2 isoform of pyruvate kinase (PKM2) in macrophages and increased its nuclear translocation, thereby promoting M1 polarization and secretion of proinflammatory M1 cytokines; this led, ultimately, to hepatocyte steatosis via paracrine effects. Taken together, these findings show that HSPA12A acts as a novel regulator of M1 macrophage polarization and NASH pathogenesis by increasing nuclear PKM2. Strategies that inhibit macrophage HSPA12A might be a potential therapeutic intervention for NASH.