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.

HSPA12A stimulates "Smurf1-Hif1α-aerobic glycolysis" axis to promote proliferation of renal tubular epithelial cells after hypoxia/reoxygenation injury.

Proliferation of renal tubular epithelial cells (TECs) is critical for the recovery after kidney ischemia/reperfusion (KI/R). However, there is still a lack of ideal therapies for promoting TEC proliferation. Heat shock protein A12A (HSPA12A) shows abundant expression in kidney in our previous studies. To investigate the role of HSPA12A in TEC proliferation after KI/R, an in vitro KI/R model was simulated by hypoxia (12 h) and reoxygenation (12 h) in human kidney tubular epithelial HK-2 cells. We found that, when hypoxia/reoxygenation (H/R) triggered HK-2 cell injury, HSPA12A expression was downregulated, and extracellular lactate, the readout of glycolysis, was also decreased. Loss and gain of functional studies showed that HSPA12A did not change cell viability after hypoxia but increased cell proliferation as well as glycolytic flux of HK-2 cells after H/R. When blocking glycolysis by 2-deoxy-D-glucose or oxamate, the HSPA12A promoted HK-2 cell proliferation was also abolished. Further analysis revealed that HSPA12A overexpression increased hypoxia-inducible factor 1α (Hif1α) protein expression and nuclear localization in HK-2 cells in response to H/R, whereas HSPA12A knockdown showed the opposite effects. Notably, pharmacological inhibition of Hif1α with YC-1 reversed the HSPA12A-induced increases of both glycolytic flux and proliferation of H/R HK-2 cells. Moreover, the HSPA12A increased Hif1α protein expression was not via upregulating its transcription but through increasing its protein stability in a Smurf1-dependent manner. The findings indicate that HSPA12A might serve as a promising target for TEC proliferation to help recovery after KI/R.

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.

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.

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.

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.

Cardiomyocyte-specific deficiency of HSPB1 worsens cardiac dysfunction by activating NFκB-mediated leucocyte recruitment after myocardial infarction.

Aims: Inadequate healing after myocardial infarction (MI) leads to heart failure and fatal ventricular rupture, while optimal healing requires timely induction and resolution of inflammation. This study tested the hypothesis that heat shock protein B1 (HSPB1), which limits myocardial inflammation during endotoxemia, modulates wound healing after MI. Methods and results: To test this hypothesis, cardiomyocyte-specific HSPB1 knockout (Hspb1-/-) mice were generated using the Cre-LoxP recombination system. MI was induced by ligation of the left anterior descending coronary artery in Hspb1-/- and wild-type (WT) littermates. HSPB1 was up-regulated in cardiomyocytes of WT animals in response to MI, and deficiency of cardiomyocyte HSPB1 increased MI-induced cardiac rupture and mortality within 21 days after MI. Serial echocardiography showed more aggravated remodelling and cardiac dysfunction in Hspb1-/- mice than in WT mice at 1, 3, and 7 days after MI. Decreased collagen deposition and angiogenesis, as well as increased MMP2 and MMP9 activity, were also observed in Hspb1-/- mice compared with WT controls after MI, using immunofluorescence, polarized light microscopy, and zymographic analyses. Notably, Hspb1-/- hearts exhibited enhanced and prolonged leucocyte infiltration, enhanced expression of inflammatory cytokines, and enhanced TLR4/MyD88/NFκB activation compared with WT controls after MI. In-depth molecular analyses in both mice and primary cardiomyocytes demonstrated that cardiomyocyte-specific knockout of HSPB1 increased nuclear factor-κB (NFκB) activation, which promoted the expression of proinflammatory mediators. This led to increased leucocyte recruitment, thereby to excessive inflammation, ultimately resulting in adverse remodelling, cardiac dysfunction, and cardiac rupture following MI. Conclusion: These data suggest that HSPB1 acts as a negative regulator of NFκB-mediated leucocyte recruitment and the subsequent inflammation in cardiomyocytes. Cardiomyocyte HSPB1 is required for wound healing after MI and could be a target for myocardial repair in MI patients.