Phosphorylation Of HSP27 Research Articles

MicroRNA216b mediated downregulation of HSP27/STAT3/AKT signaling is critically involved in lambertianic acid induced apoptosis in human cervical cancers.

Since heat shock protein (HSP27) is a prognostic marker in cervical cancer, in the present study, the apoptotic mechanism of lambertianic acid (LA) was investigated in human cervical cancers in association with HSP27/STAT3/AKT signaling axis. LA exerted significant cytotoxicity, induced sub-G1 population, and increased the cleavage of Poly (ADP-ribose) polymerase (PARP) and cysteine aspartyl-specific protease 3 (caspase3) in HeLa and Caski cancer cells. Consistently, LA downregulated anti-apopotic genes such as B-cell lymphoma 2 (Bcl-2) and inhibitors of apoptosis proteins (c-IAP) in HeLa and Caski cells. Furthermore, LA-inhibited phosphorylation of HSP27, signal transducer, and activator of transcription 3 (STAT3) and Protein kinase B (AKT) through disturbing the binding of HSP27 with STAT3 or AKT in HeLa cells. Notably, LA upregulated the level of miR216b in HeLa and Caski cells. Consistently, miR216b mimic suppressed phosphorylation of HSP27 and reduced the expression of pro-PARP, while miR216b inhibitor reversed the ability of LA to attenuate phosphorylation of AKT, HSP27, and STAT3 and to reduce the expression of pro-PARP in HeLa cells. Overall, our findings suggest that miRNA216b mediated inhibition of HSP27/STAT3/ AKT signaling axis is critically involved in LA-induced apoptosis in cervical cancers.

Reduced LINC00551 expression promotes proliferation and invasion of esophageal squamous cancer by increase in HSP27 phosphorylation

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers, and long noncoding RNAs (lncRNAs) regulate gene expression or activities. This study investigated the role of lncRNA LINC00551 in ESCC development and progression. Three paired ESCC and normal tissues were subjected to next-generation sequencing and we identified 82 upregulated and 60 downregulated lncRNAs, including LINC00551, which was confirmed to markedly downregulated in 78 ESCC tissues and in the Gene Expression Profiling Interactive Analysis data set. Downregulated LINC00551 expression was associated with lymph node metastasis, advanced TNM stage, and tumor size. Moreover, downregulated LINC00551 expression was also associated with poor progression-free survival and overall survival of ESCC patients. In vitro and in vivo, LINC00551 overexpression inhibited ESCC cell proliferation and invasion, whereas knockdown of LINC00551 expression promoted ESCC cell proliferation and invasion. RNA pull-down and mass spectrometry assays identified the potential LINC00551 binding proteins, and HSP27 was a promising LINC00551 targeting proteins after RNA immunoprecipitation assay. At the protein level, LINC00551 bound to and decreased HSP27 phosphorylation, and in turn, downregulated ESCC cell proliferation and invasion. The current study demonstrated the functional significance of LINC00551 in ESCC development, progression, and prognosis. Further study will assess LINC00551 as a novel prognostic marker or therapeutic target for ESCC.

HSP27 regulates viability and migration of cancer cell lines following irradiation

Despite improvements of radiotherapy and better outcomes of cancer patients resistances still limit the therapeutic success. The combined treatment of tumors by the use of irradiation as well as targeted therapies is a promising approach. By the use of a proteomic screening of lung and head and neck cancer cell lines we identified the heat shock protein HSP27 as a potential target protein for a combined treatment strategy.Overall expression of HSP27 was distinctly lower in HNSCCUM-02T cells which have a high HSP27 phosphorylation ratio, whereas A549 cells revealed the opposite. Irradiation and inhibition of HSP27 phosphorylation by MKII inhibition resulted in a significantly reduced viability in both cell lines. While irradiation impaired migration only in HNSCCUM-02T cells, MKII inhibition exerted that effect in both cell lines. In contrast, knockdown of HSP27 compromised the viability only in A549 cells. Additionally, MKII inhibition counteracts radiation-induced phosphorylation of HSP27 which causes an additive toxicity and reduced migratory capacity in HNSCCUM-02T when combined.Inhibition of HSP27 expression and phosphorylation in combination with radiotherapy may be an effective treatment option to overcome resistances.

ZNF322A-mediated protein phosphorylation induces autophagosome formation through modulation of IRS1-AKT glucose uptake and HSP-elicited UPR in lung cancer

BackgroundZNF322A is an oncogenic transcription factor that belongs to the Cys2His2-type zinc-finger protein family. Accumulating evidence suggests that ZNF322A may contribute to the tumorigenesis of lung cancer, however, the ZNF322A-mediated downstream signaling pathways remain unknown.MethodsTo uncover ZNF322A-mediated functional network, we applied phosphopeptide enrichment and isobaric labeling strategies with mass spectrometry-based proteomics using A549 lung cancer cells, and analyzed the differentially expressed proteins of phosphoproteomic and proteomic profiles to determine ZNF322A-modulated pathways.ResultsZNF322A highlighted a previously unidentified insulin signaling, heat stress, and signal attenuation at the post-translational level. Consistently, protein-phosphoprotein-kinase interaction network analysis revealed phosphorylation of IRS1 and HSP27 were altered upon ZNF322A-silenced lung cancer cells. Thus, we further investigated the molecular regulation of ZNF322A, and found the inhibitory transcriptional regulation of ZNF322A on PIM3, which was able to phosphorylate IRS1 at serine1101 in order to manipulate glucose uptake via the PI3K/AKT/mTOR signaling pathway. Moreover, ZNF322A also affects the unfolded protein response by phosphorylation of HSP27S82 and eIF2aS51, and triggers autophagosome formation in lung cancer cells.ConclusionsThese findings not only give new information about the molecular regulation of the cellular proteins through ZNF322A at the post-translational level, but also provides a resource for the study of lung cancer therapy.

Focal adhesion kinase is activated by microtubule-depolymerizing agents and regulates membrane blebbing in human endothelial cells.

Microtubule‐depolymerizing agents can selectively disrupt tumor vessels via inducing endothelial membrane blebbing. However, the mechanism regulating blebbing is largely unknown. IMB5046 is a newly discovered microtubule‐depolymerizing agent. Here, the functions of focal adhesion kinase (FAK) during IMB5046‐induced blebbing and the relevant mechanism are studied. We found that IMB5046 induced membrane blebbing and reassembly of focal adhesions in human vascular endothelial cells. Both FAK inhibitor and knock‐down expression of FAK inhibited IMB5046‐induced blebbing. Mechanism study revealed that IMB5046 induced the activation of FAK via GEF‐H1/ Rho/ ROCK/ MLC2 pathway. cRGD peptide, a ligand of integrin, also blocked IMB5046‐induced blebbing. After activation, FAK further promoted the phosphorylation of MLC2. This positive feedback loop caused more intensive actomyosin contraction and continuous membrane blebbing. FAK inhibitor blocked membrane blebbing via inhibiting actomyosin contraction, and stimulated stress fibre formation via promoting the phosphorylation of HSP27. Conclusively, these results demonstrate that FAK is a molecular switch controlling endothelial blebbing and stress fibre formation. Our study provides a new molecular mechanism for microtubule‐depolymerizing agents to be used as vascular disrupting agents.

Cardiac Protective Role of Heat Shock Protein 27 in the Stress Induced by Drugs of Abuse

Heat shock proteins (HSP) are induced after different stress situations. Some of these proteins, particularly HSP-27, function as markers to indicate cellular stress or damage and protect the heart during addictive processes. Morphine withdrawal induces an enhancement of sympathetic activity in parallel with an increased HSP-27 expression and phosphorylation, indicating a severe situation of stress. HSP-27 can interact with different intracellular signaling pathways. Propranolol and SL-327 were able to antagonize the activation of hypothalamic-pituitary adrenal (HPA) axis and the phosphorylation of HSP-27 observed during morphine withdrawal. Therefore, β-adrenergic receptors and the extracellular signal-regulated kinase (ERK) pathway would be involved in HPA axis activity, and consequently, in HSP-27 activation. Finally, selective blockade of corticotrophin releasing factor (CRF)-1 receptor and the genetic deletion of CRF1 receptors antagonize cardiac adaptive changes. These changes are increased noradrenaline (NA) turnover, HPA axis activation and decreased HSP-27 expression and phosphorylation. This suggests a link between the HPA axis and HSP-27. On the other hand, morphine withdrawal increases µ-calpain expression, which in turn degrades cardiac troponin T (cTnT). This fact, together with a co-localization between cTnT and HSP-27, suggests that this chaperone avoids the degradation of cTnT by µ-calpain, correcting the cardiac contractility abnormalities observed during addictive processes. The aim of our research is to review the possible role of HSP-27 in the cardiac changes observed during morphine withdrawal and to understand the mechanisms implicated in its cardiac protective functions.

Modulation of Titin-Based Stiffness in Hypertrophic Cardiomyopathy via Protein Kinase D

The giant protein titin performs structure-preserving functions in the sarcomere and is important for the passive stiffness (Fpassive) of cardiomyocytes. Protein kinase D (PKD) enzymes play crucial roles in regulating myocardial contraction, hypertrophy, and remodeling. PKD phosphorylates myofilament proteins, but it is not known whether the giant protein titin is also a PKD substrate. Here, we aimed to determine whether PKD phosphorylates titin and thereby modulates cardiomyocyte Fpassive in normal and failing myocardium. The phosphorylation of titin was assessed in cardiomyocyte-specific PKD knock-out mice (cKO) and human hearts using immunoblotting with a phosphoserine/threonine and a phosphosite-specific titin antibody. PKD-dependent site-specific titin phosphorylation in vivo was quantified by mass spectrometry using stable isotope labeling by amino acids in cell culture (SILAC) of SILAC-labeled mouse heart protein lysates that were mixed with lysates isolated from hearts of either wild-type control (WT) or cKO mice. Fpassive of single permeabilized cardiomyocytes was recorded before and after PKD and HSP27 administration. All-titin phosphorylation was reduced in cKO compared to WT hearts. Multiple conserved PKD-dependent phosphosites were identified within the Z-disk, A-band and M-band regions of titin by quantitative mass spectrometry, and many PKD-dependent phosphosites detected in the elastic titin I-band region were significantly decreased in cKO. Analysis of titin site-specific phosphorylation showed unaltered or upregulated phosphorylation in cKO compared to matched WT hearts. Fpassive was elevated in cKO compared to WT cardiomyocytes and PKD administration lowered Fpassive of WT and cKO cardiomyocytes. Cardiomyocytes from hypertrophic cardiomyopathy (HCM) patients showed higher Fpassive compared to control hearts and significantly lower Fpassive after PKD treatment. In addition, we found higher phosphorylation at CaMKII-dependent titin sites in HCM compared to control hearts. Expression and phosphorylation of HSP27, a substrate of PKD, were elevated in HCM hearts, which was associated with increased PKD expression and phosphorylation. The relocalization of HSP27 in HCM away from the sarcomeric Z-disk and I-band suggested that HSP27 failed to exert its protective action on titin extensibility. This protection could, however, be restored by administration of HSP27, which significantly reduced Fpassive in HCM cardiomyocytes. These findings establish a previously unknown role for PKDin regulating diastolic passive properties of healthy and diseased hearts.

Small GTPase Cdc42 and p130 Crk‐associated substrate (p130Cas) contribute to actin cytoskeleton dynamics in the myogenic response of rat cerebral arteries to intraluminal pressure

The constriction and relaxation of cerebral resistance arteries/arterioles in response to changes in intraluminal pressure, referred to as the myogenic response, is a key physiological mechanism for the regulation of blood pressure and flow in the brain. Actin cytoskeleton dynamics in vascular smooth muscle cells involving increased actin polymerization contribute to myogenic tone development in response to pressure elevation, but our understanding of the cellular mechanisms involved is limited. Members of the Rho family of small GTPases including RhoA, Rac1 and Cdc42 are established regulators of actin cytoskeleton dynamics. RhoA‐Rho‐associated coiled‐coil kinase (ROCK)‐mediated signaling contributes to actin polymerization in the cerebral arterial myogenic response, but whether Rac1 and/or Cdc42 play a role is not known. Here, the contribution of Rac1 and Cdc42 to myogenic vasoconstriction of endothelium‐denuded posterior cerebral arteries of adult rats was assessed by pressure myography combined with ultra‐sensitive immunoblotting to quantify G‐actin content, and level of phosphorylation of myosin regulatory light chain (LC20), myosin phosphatase targeting subunit 1 (MYPT1), p130Cas, N‐WASP, Arp2, cofilin and HSP27 in the absence and presence of GTPase inhibition. Treatment of vessels with Rac1 inhibitor Ehop‐016 (2–4 μM) before or after pressurization from 10 to 80 mmHg had no effect, whereas inhibition of Cdc42 with ML‐141 (1–10 μM) suppressed myogenic tone development. Significantly, ML‐141 (10 μM) did not affect the increase in LC20 or MYPT1‐T855 phosphorylation in response to pressurization from 10 to 80 mmHg, but it completely blocked the 50% decline in G‐actin content. Pressurization increased the level of p130Cas (Y165, Y249, Y410), N‐WASP (Y256), Arp2 (T237/238), HSP27 (S82) and cofilin (S3) phosphorylation. ML‐141 blocked the increase in p130Cas, N‐WASP, Arp2 and HSP27, but not cofilin phosphorylation. In contrast, inhibition of ROCK activity with H1152 (0.3 μM), which is known to suppress MYPT1, LC20 and cofilin phosphorylation, had no effect on the level of p130Cas, N‐WASP or Arp2 phosphorylation. Pharmacological suppression of alpha5‐integrins‐, focal adhesion kinase‐, or Src kinase‐dependent signaling, that inhibit MYPT1 and LC20 phosphorylation, G‐actin reduction and myogenic tone development, also prevented the pressure‐dependent increase in p130Cas, N‐WASP, Arp2, HSP27 and cofilin phosphorylation. Taken together, these data suggest that the myogenic response of rat cerebral arteries involves Cdc42 signaling that stimulates actin cytoskeleton dynamics via a p130Cas‐, N‐WASP‐ and Arp2‐dependent mechanism. This pathway is distinct from RhoA‐ROCK signaling that increases crossbridge cycling via increased MYPT1 and LC20 phosphorylation, as well as actin dynamics via cofilin. Activation of both RhoA and Cdc42 signaling in response to pressure elevation is dependent on integrin, focal adhesion kinase and Src kinase activity.Support or Funding InformationThis work was funded by a research grant from the Canadian Institutes of Health Research MOP‐97988 (WCC).

The Implications of TLK1‐MK5 Signaling Axis in Prostate Cancer Cell Motility and Invasion

BackgroundProstate cancer (PCa) is the 2nd leading death‐causing malignancy in men where the majority of fatalities occur due to metastasis of cancer cells to adjacent and distal organs. Devising a therapeutic intervention requires the identification of underlying signaling pathways involved in the progression of PCa. We identified the novel interaction between two kinases that may initiate a signaling cascade promoting metastasis. One kinase is MAPK activated protein kinase 5 (MAPKAPK5 or MK5) which plays a debated role in cellular motility along with other cellular processes. Our observations are that MK5 interacts with another S/T kinase, called tousled like kinase 1 (TLK1), which may also have a potential role in cellular motility which is largely unstudied, apart from one report in breast cancer on TLK2. In PCa, TLK1‐MK5 signaling axis might be crucial as androgen deprivation therapy leads to increased expression of both TLK1 and MK5 in metastatic castrate‐resistant prostate cancer patients. Our working hypothesis is that TLK1‐MK5 axis promotes PCa cell motility and invasion through actin rearrangement as well as focal adhesion proteins modification and disruption of this interaction may help to keep the tumor localized.Method and ResultsA previous protoarray interaction study from our lab identified MK5 as one of the novel interactors of TLK1 with high confidence (p<0.00005). To validate this interaction, we overexpressed EGFP‐MK5 in HEK 293 cells and performed a reciprocal Co‐IP study targeting both TLK1 and EGFP‐MK5 that confirmed their interaction in vivo. Additionally, a reciprocal His‐ and GST‐pull down assays demonstrated the direct and physical interaction between TLK1 and MK5 in vitro. Another Co‐IP experiment revealed that TLK1‐MK5 interaction starts with the attachment of the cells to the ECM. We conducted several scratch wound repair assays to find out if TLK1‐MK5 axis enhances the migration rate of the cells. Inhibition of the kinase activity of TLK1 by using a specific inhibitor (thioridazine) decreases the migration rate of the HEK 293 cells, which supports the importance of TLK1 function in motility. Wound repair was also demonstrated to be faster in overexpressed EGFP‐MK5 in LNCaP and MEF cells and compared also to MK5 knock out cells. An in‐vitro Kinase assay using [y‐32P] ATP demonstrated that incubation of purified TLK1B and purified MK5 increases the phosphorylation of MK5, but not on TLK1B. Another in‐vitro kinase assay using HSP27 as a substrate of MK5 revealed that TLK1B increases the kinase activity of MK5 towards HSP27 phosphorylation. Mass spectrometric analysis identified three unique phosphorylation sites in MK5 (S160, S354, S386) by TLK1B which are mapped to the kinase, NLS/NES and ERK3 binding domain of MK5. We also measured the protein level of several focal adhesion proteins in TLK1 and MK5 co‐expressed HEK 293 cells such as total FAK, phospho‐FAK (Tyr397), total Paxillin, Phospho‐Paxillin (Tyr118, Ser178), etc. and observed activating phosphorylation.ConclusionOur data support that TLK1‐MK5 interaction is functionally involved in cell motility and migration and hence, disruption of this axis may inhibit the metastasis of PCa.Support or Funding InformationDoD‐PCRP grant W81XWH‐17‐1‐0417

Heat stress defense in dental pulp stem cells

Many dental treatment procedures can generate excessive heat within a short period of time. The heat on the tooth surface can be transduced to the dental pulp chamber and lead to alteration of dental pulp stem cells, vascular structures and sensory neurons within the chamber. It has been reported that elevation of temperature by 5.5°C in the pulp chamber caused 15% irreversible pulpal damage. However, the molecular changes of dental pulp cells post heat stress has not been characterized. In this study, we explored the changes in human dental pulp Stem Cells from the Exfoliated Deciduous teeth (SHED) after exposure to heat stress.Lysate of SHED cells were collected after exposure to heat stress at 42°C or normal culture temperature at 37°C. Western blots were used to examine the expression of Heat Shock Proteins (HSP). Human cytokine arrays (AAH‐CYT‐5, RayBiotech) were used to evaluate the changes in the expression of 80 human cytokines in SHED cells after heat stress.Short term exposure to heat stress induced phosphorylation of HSP27. Prolonged exposure further increased expression of other heat shock proteins, which have been reported to play an important role in cell protection under stress. In addition, transient exposure to heat stress caused differential expression of cytokines in SHED cells, suppressing expression of cytokines as IL‐1a and IL‐10, while increasing expression of glial cell derived neurotrophic factor (GDNF) and insulin‐like growth factor ‐1 (IGF‐1).The results showed HSPs and multiple cytokines play important role in detecting and responding to heat stress on the SHED cells. These factors could potentially protect SHED cells from thermal damage and promote stem cell regeneration post thermal damage.Support or Funding InformationFaculty Scholarly/Artistic Activities Award DDNA12‐013 University of the Pacific.

Hsp27 chaperones FUS phase separation under the modulation of stress-induced phosphorylation.

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid-liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.

Dioscin facilitates ROS-induced apoptosis via the p38-MAPK/HSP27-mediated pathways in lung squamous cell carcinoma.

Lung squamous cell carcinoma (SCC) is one of the deadliest cancers both in China and worldwide. To date, the efficacy of lung SCC treatments is limited. Recent studies have elucidated the powerful anti-tumour role of dioscin in different human cancers. Here, our study aims to investigate the effect of dioscin on lung SCC and its underlying mechanism. First, we found that dioscin not only inhibited cell proliferation and cell migration and induced cell apoptosis in lung SCC cells but also suppressed tumour growth in tumour-bearing mice. Furthermore, we noted that the accumulation of intracellular reactive oxygen species (ROS) was triggered by dioscin in lung SCC cells, leading to the phosphorylation of HSP27 through p38-MAPK and consequent cell apoptosis. The activation of p38-MAPK/HSP27 induced by the p38-MAPK activator Anisomycin enhanced the apoptosis of lung SCC cells, while the ROS inhibitor N-acetyl-L-cysteine (NAC) and the p38-MAPK inhibitor SB203580 both attenuated dioscin-mediated cell apoptosis. Moreover, NAC suppressed the activation of p38-MAPK/HSP27 that induced by dioscin. In conclusion, these results confirm that dioscin facilitates ROS-induced apoptosis via the p38-MAPK/HSP27-mediated pathway in lung SCC.

T-cadherin inhibits invasion and migration of endometrial stromal cells in endometriosis.

What is the expression level of T-cadherin in endometriosis, and does T-cadherin play a role in regulating invasion and migration of endometrial stromal cells? T-cadherin expression was reduced in ectopic endometriotic lesions compared to eutopic endometrium, and T-cadherin overexpression inhibited the invasion and migration of endometrial stromal cells. Endometriosis is a disease that involves active cell invasion and migration. T-cadherin can inhibit cell invasion, migration and proliferation in various cancer cells, but its role in endometriosis has not been investigated. We explored the expression status of T-cadherin in 40 patients with and 24 without endometriosis. We also isolated endometrial stromal cells to study the invasion, migration and signaling pathway regulation of T-cadherin overexpression. Patients were recruited at the Guangzhou Women and Children's Medical Center to study the expression levels of T-cadherin. The expression of T-cadherin was detected by immunohistochemistry staining and western blot. H-score was used to evaluate the staining intensity of T-cadherin. The correlation between T-cadherin expression levels (H-score) and endometriosis patients' age, stage, lesion size and adhesion was analyzed. Endometrial stromal cells from patients with and without endometriosis were isolated, and cell invasion and migration were detected by transwell assays after T-cadherin overexpression. The expression of vimentin in T-cadherin-overexpressed cells was detected by western blot. After T-cadherin overexpression, the phosphorylation profile of signaling pathway proteins was detected with the Proteome Profiler Human Phospho-Kinase Array Kit. There was no difference in the expression of T-cadherin in the normal endometrium of control patients and the eutopic endometrium of endometriotic patients, but it was significantly decreased in the ectopic endometrium of endometriotic patients, compared with control endometrium and eutopic endometrium of endometriosis patients (P< 0.0001, for both). Western blot analysis also showed that the expression of T-cadherin was decreased in ectopic endometriotic lesions, but not the normal control endometrium or the endometriotic eutopic endometrium. The results of transwell assays indicated that T-cadherin overexpression inhibited the invasion and migration of endometrial stromal cells. In addition, T-cadherin overexpression promoted the phosphorylation of HSP27 (S78/S82) and JNK 1/2/3 (T183/Y185, T221/Y223) and decreased the expression of vimentin, MMP2 and MMP9 in eutopic endometriosis stromal cells. N/A. The control group were patients with benign gynecological conditions (e.g. uterus myoma, endometrial or cervical polyp), which may have genetic or epigenetic variations associated with T-cadherin expression and signaling pathways. The case numbers of involved endometriosis and control patients were limited. This study only used endometrial stromal cells from patients with or without endometriosis. Ideally, ectopic endometrial stromal cells of the ovarian endometriotic lesions should also be utilized to explore the function of T-cadherin. Further investigation of the role of T-cadherin in endometriosis may generate new potential therapeutic targets for this complex disorder. This study was supported by the Natural Science Foundation of Guangdong Province (2016A030313495), National Natural Science Foundation of China (81702567, 81671406, 31871412), the Science and Technology Programs of Guangdong (2017A050501021), Medical Science Technology Research Fund of Guangdong Province (A2018075), the Science and Technology Programs of Guangzhou City (201704030103), Internal Project of Family Planning Research Institute of Guangdong Province (S2018004), Post-doc initiation fund of Guangzhou (3302) and Post-doc science research initiation fund of Guangzhou Women and Children's Medical Center (20160322). There are no conflicts of interest.

Ivermectin inhibits HSP27 and potentiates efficacy of oncogene targeting in tumor models.

HSP27 is highly expressed in, and supports oncogene addiction of, many cancers. HSP27 phosphorylation is a limiting step for activation of this protein and a target for inhibition, but its highly disordered structure challenges rational structure-guided drug discovery. We performed multistep biochemical, structural, and computational experiments to define a spherical 24-monomer complex composed of 12 HSP27 dimers with a phosphorylation pocket flanked by serine residues between their N-terminal domains. Ivermectin directly binds this pocket to inhibit MAPKAP2-mediated HSP27 phosphorylation and depolymerization, thereby blocking HSP27-regulated survival signaling and client-oncoprotein interactions. Ivermectin potentiated activity of anti-androgen receptor and anti-EGFR drugs in prostate and EGFR/HER2-driven tumor models, respectively, identifying a repurposing approach for cotargeting stress-adaptive responses to overcome resistance to inhibitors of oncogenic pathway signaling.

The release of phosphorylated-HSP27 from activated platelets of obstructive sleep apnea syndrome (OSAS) patients

Obstructive sleep apnea syndrome (OSAS) is a well known risk of arterial thrombosis that results in cardiovascular morbidity. It has been reported that platelet aggregability is enhanced in patients with OSAS. In the present study, we investigated whether phosphorylated-HSP27 is released from the activated platelets of OSAS patients. Patients diagnosed with OSAS (n=21) were recruited, and platelet-rich plasma (PRP) was stimulated by ADP, ristosetin, collagen, and thrombin receptor-activating peptide. Platelet aggregation was measured using an aggregometer with a laser-scattering system. The levels of protein phosphorylation and the released levels of phosphorylated-HSP27 were determined by Western blot analysis and an ELISA, respectively. The phosphorylation of HSP27 in the platelets was induced by the stimulators. The released levels of phosphorylated-HSP27 was correlated with the levels of phosphorylated-HSP27 stimulated by ADP or collagen. The levels of ADP-induced phosphorylated-HSP27 were correlated with those of both phosphorylated-protein kinase B (Akt) and phosphorylatd-p38 mitogen-activated protein kinase; however, the levels of phosphorylated-HSP27 stimulated by collagen were correlated with phosphorylated-Akt levels only. The ED50 value of ADP on the platelet aggregation in OSAS (1.067±0.128μM) was lower than that in healthy subjects (1.778±0.122μM) and was inversely correlated with both the value of minimum SpO2 and the released level of phosphorylated-HSP27 stimulated by ADP. The results strongly suggest that phosphorylated-HSP27 is released from the activated platelets of OSAS patients.

Nanometric targeting of type 9 adenylyl cyclase in heart.

Adenylyl cyclases (ACs) convert ATP into the classical second messenger cyclic adenosine monophosphate (cAMP). Cardiac ACs, specifically AC5, AC6, and AC9, regulate cAMP signaling controlling functional outcomes such as heart rate, contractility and relaxation, gene regulation, stress responses, and glucose and lipid metabolism. With so many distinct functional outcomes for a single second messenger, the cell creates local domains of cAMP signaling to correctly relay signals. Targeting of ACs to A-kinase anchoring proteins (AKAPs) not only localizes ACs, but also places them within signaling nanodomains, where cAMP levels and effects can be highly regulated. Here we will discuss the recent work on the structure, regulation and physiological functions of AC9 in the heart, where it accounts for <3% of total AC activity. Despite the small contribution of AC9 to total cardiac cAMP production, AC9 binds and regulates local PKA phosphorylation of Yotiao-IKs and Hsp20, demonstrating a role for nanometric targeting of AC9.

Infusion of Mesenchymal Stem Cells Combined with Epithelial Progenitor Cells Promotes Injured Intestinal Repairing after Hematopoietic Cell Transplantation

Pre-transplant chemoradiotherapy can impair intestinal barrier function and disrupt immune homeostasis, and thus increase the incidence of infection and other related complications. Mesenchymal stem cells (MSCs) have the potential to rescue Inflammatory Bowel Disease and intestinal graft versus host disease owing to their immunosuppressive capabilities. However, limited studies regarding MSC administration has been reported to aggravate T cell-mediated tissue injury. Plus, endothelial progenitor cells (EPCs) could improve endothelium repair, facilitate hematopoietic reconstitution and alleviate complications associated with hematopoietic cell transplantation (HCT). The key goals of this study were to i) identify the role of MSC derived soluble and contact dependent factors and how these affect intestinal injury; ii) investigate the effect of MSC combined EPC therapy for repairing the injured intestine. In this study, BALB/c mice were randomly divided into five groups, namely, total body irradiation only, bone marrow transplantation (BMT), MSC(BMT with 1 × 106MSC infusion), EPC(BMT with 5 × 105 EPCs infusion), and MSC+EPC (BMT with 1 × 106 MSC and 5 × 105 EPCs infusion). Results showed that the best performance of intestine was found in the MSC+EPC treated group, which showed more epithelial and goblet cells, and less apoptosis cells and adjacent crypts fusion in intestine morphology. EPC or MSC only group improved the intestinal injuries slightly compared with BMT group. The higher MECA-32 expression level was found in the intestinal tissue of MSC+EPC and MSC groups compared with other treated groups. The tight junction molecules occludin had highest expression level in the intestinal epithelial cells of MSC+EPC group, followed by MSC group and then EPC groups, but the expression level in BMT group was extremely low. Further study demonstrated that in MSC+EPC group the phosphorylated P38 enhanced heat shock protein HSP27 activation, which promoted cytoskeleton reconstruction and intestinal epithelial cells proliferation; and phosphorylated P38 also down-regulated the expression of apoptosis-related molecule caspase3. Moreover, the multiple effects of MSCs on cellular immunity may reflect their diverse influences on the different T-cell subpopulations. MSC+EPC infusion increased the number of IL-17A secreting cells (Th17 and Tc17) in mesenteric lymph nodes early after HCT, and decreased Th1 cells at day 10 and delayed expanding of Tc1 from day 10 to day 15. The soluble IL-17A in intestinal tissue was significantly increased after MSC+EPC infusion in according with IL-17A secreting cells in mesenteric lymph nodes. Furthermore, the gut bacterial information analyzed by high throughput sequencing uncovered that MSC and MSC+EPC groups had higher bacterial diversity and richness compared with other groups, and the bacterial community structures exhibited big changes regarding different treatment. Co-occurrence analysis demonstrated that the genus Akkermansia inducing PD-1 expression of T cells had significant correlation with phosphorylation of P38 and HSP27 in MSC+EPC and EPC treated groups, which indicated Akkermansia may be a key bacteria participanting the intestinal repairing in MSC and MSC+EPC groups. In conclusion, this study confirmed that the MSC+EPC infusion can obviously repair injured intestine, and gut bacteria Akkermansia correlated with phosphorylation of P38 and HSP27 expression and participated in intestinal repairing. Disclosures No relevant conflicts of interest to declare.

Millimeter-wave pulsed heating in vitro: cell mortality and heat shock response

Millimeter wave (MMW)-induced heating represents a promising alternative for non-invasive hyperthermia of superficial skin cancer, such as melanoma. Pulsed MMW-induced heating of tumors allows for reaching high peak temperatures without overheating surrounding tissues. Herein, for the first time, we evaluate apoptotic and heat shock responses of melanoma cells exposed in vitro to continuous (CW) or pulsed-wave (PW) amplitude-modulated MMW at 58.4 GHz with the same average temperature rise. Using an ad hoc exposure system, we generated 90 min pulse train with 1.5 s pulse duration, period of 20 s, amplitude of 10 °C, and steady-state temperature at the level of cells of 49.2 °C. The activation of Caspase-3 and phosphorylation of HSP27 were investigated using fluorescence microscopy to monitor the spatial variation of cellular response. Our results demonstrate that, under the considered exposure conditions, Caspase-3 activation was almost 5 times greater following PW exposure compared to CW. The relationship between the PW-induced cellular response and SAR-dependent temperature rise was non-linear. Phosphorylation of HSP27 was 58% stronger for PW compared to CW. It exhibits a plateau for the peak temperature ranging from 47.7 to 49.2 °C. Our results provide an insight into understanding of the cellular response to MMW-induced pulsed heating.

Phosphorylated Hsp27 prevents LPS-induced excessive inflammation in THP-1 cells via suppressing ROS-mediated upregulation of CBP.

Heat shock protein 27 (Hsp27) is a member of the small heat shock protein family expressed at high levels to protect cells against heat shock and other conditions of stress. Hsp27 has been indicated in the regulation of inflammation signaling pathway, and Hsp27 phosphorylation is vital for efficient control of host-defense response in early stages of lipopolysaccharide (LPS)-stimulated inflammation. The notion that CREB-binding protein (CBP) is involved in the regulation of two major transcription factors, nuclear factor-κB (NF-κB) and AP-1, suggests that CBP, as a coactivator protein, may also play an important role in the cellular response to inflammation. Here, we explored the mechanism underlying the regulatory relationships between Hsp27 and CBP in THP-1 cells, and found that phosphorylated Hsp27 was critical to the protein level of CBP. Furthermore, in exploring the signaling mechanisms underlying its action, we found that p38MAPK-MK2-Hsp27 regulated NF-κB via CBP, which acted as a multi-protein complex assembly scaffold. Finally, we demonstrated that phosphorylated Hsp27 reduced reactive oxygen species accumulation thereby significantly repressed LPS-induced excessive increase of CBP. Taken together, our data demonstrated that Hsp27, in its phosphorylation state, plays a critical role in controlling LPS-induced inflammatory response by modulating CBP.

Phosphorylated Heat Shock Protein 27 Inhibits Lipopolysaccharide-Induced Inflammation in Thp1 Cells by Promoting TLR4 Endocytosis, Ubiquitination, and Degradation

The aims of this study were to investigate the effect of Hsp27 on LPS-induced inflammation and identify the precise mechanisms about how Hsp27 regulates LPS-induced TLR4 signaling in Thp1 cells. Thp1 cells were transfected with Flag-Hsp27 or pcDNA3.1, and then treated with LPS for indicated time. TNF-α, IL-1β, and IL-6 were determined by ELISA. The protein levels of Hsp27, p-Hsp27 (Ser15, Ser78, and Ser82), and TLR4 were measured by Western blotting. In vitro study showed that over-expression of Hsp27 downregulated the release of TNF-α, IL-1β, and IL-6 and suppressed the activation of TLR4 signals after stimulated by LPS. The location of TLR4 and RAB5 was detected by confocal microscopy. Immunoprecipitation was used to determine the ubiquitination and degradation of TLR4 and interaction between Hsp27 and TLR4. Results showed that Hsp27 could promote TLR4 endocytosis and ubiquitination and degradation. Further research revealed that Hsp27 was phosphorylated after LPS, only phosphorylated Hsp27 can interact with TLR4 and inhibit the activation of TLR4 signaling, which was demonstrated by inhibition of Hsp27 phosphorylation with inhibitors or transfection of Hsp27 mutants into Thp1 cells. Phosphorylated Hsp27 reduced the release of TNF-α, IL-1β, and IL-6, and suppressed the activation of TLR4 signaling by promoting TLR4 endocytosis, ubiquitination, and degradation.