Effect Of Heat Shock Protein 27 Research Articles

Effect of HSP27 and Cofilin in the injury of hypoxia/reoxygenation on hepatocyte membrane F-actin microfilaments

Hypoxia–reoxygenation (H/R) injury hepatocyte models were established to simulate the ischemia/reperfusion injury of transplanted organ. Through the study of the molecular mechanism of H/R on the F-actin damage of the liver cytomembrane, the mechanism of F-actin damage induced by ischemia and reperfusion was studied from the level of cell and molecule.The hypoxic environment of cells in vitro was simulated by chemical hypoxia agent CoCl2. Liver cells were detected by MTT, H/R group was subdivided into 3 subgroups: H/R 2, 4, and 6 h. Changes of cell shape and the growth state, apoptosis, ultrastructural changes, and the changes in F-actin microfilament content were observed. Heat shock protein 27 (HSP27), Cofilin, and F-actin gene and protein levels were determined by real-time polymerase chain reaction and western blot assay, respectively.Cells showed circular adherence growth under normal circumstances, while the spindle cells and shedding cells were significantly increased in H/R groups. Apoptosis cells in H/R group were increased significantly with the extension of hypoxia time. The number of endoplasmic reticulum was decreased significantly in the H/R group, the mitochondrion hydropic was degenerated and the glycogen was disappeared. The F-actin fibers in the H/R group were disordered, the morphology of the fibers was obviously decreased, and the fluorescence staining decreased obviously (P < .05). The transcription and expression levels of HSP27, Cofilin, and F-actin were significantly lower than those in the control group (P < .05).These results demonstrate that H/R can affect the correct assembly of F-actin microfilaments and weakens the normal cycle of F-actin microfilaments through inhibiting the protein expression and gene transcription of HSP27 and Cofilin in hepatocytes, thereby changing the skeleton of F-actin microfilaments.

The Effects of HSP27 on Gemcitabine-Resistant Pancreatic Cancer Cell Line Through Snail

To evaluate the regulation mechanism of heat shock protein 27 (HSP27) on gemcitabine (GEM) resistance of pancreatic cancer cell. The expression vectors pEGFP-C1-HSP27 and the vectors of MicroRNA targeting Snail were introduced into GEM-sensitive pancreatic cancer SW1990 cells, and the vectors of small hairpin RNA targeting HSP27 were transfected into SW1990 and GEM-resistant SW1990/GEM cells. The expressions of HSP27, p-HSP27 (Ser82), Snail, ERCC1, and E-cadherin were evaluated by Western blotting. The sensitivity of transfected cells to GEM was detected by CCK-8 assay and Annexin V-FITC apoptosis assay. As compared to SW1990, SW1990/GEM showed significantly increased expressions of HSP27, p-HSP27, Snail and ERCC1 with decreased expression of E-cadherin. By increasing HSP27 expression, we found increase of Snail and ERCC1 with reduction of E-cadherin expressions, while reduction of HSP27 expression caused reduction of Snail and ERCC1 but increase of E-cadherin expressions. Downregulation of Snail resulted in the reduction of ERCC1 expression and increase of E-cadherin. Furthermore, downregulation of HSP27 or snail caused increased GEM sensitivity of pancreatic cancer cells, and upregulation of HSP27 showed the opposite results. There is an inverse correlation between HSP27 expression and GEM sensitivity of SW1990 cells, which might be realized by regulating E-cadherin and ERCC1 expressions through Snail.

Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed-batch bioreactors.

CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures.

Abstract 16767: Exosomes Mediate Heat Shock Protein 27 Athero-Protective Signalling

Introduction: Exosomes represent a discrete population of nano-vesicles secreted by a variety of cells and may contain unique protein cargo that play a role in inter-cellular signalling. We previously showed (ATVB 2009, JACC 2013) that membrane-like vesicles mediate the release of the novel biomarker and athero-protective agent, Heat Shock Protein 27 (HSP27), however did not explore downstream signalling effects of HSP27-laden exosomes. Hypothesis: Monocyte derived exosomes facilitate the transfer of HSP27 to human aortic endothelial cells (HAEC) and THP-1 MΦ to elicit athero-protective signalling effects. Methods: Exosome particles were purified from conditioned medium of THP-1 monocytes. As we previously showed that HSP27 athero-protection involves activation of NF-kB (e.g., with resultant changes in the expression of key athero-protective genes) we sought to determine if exosomes alter NF-kB signalling, as well as augment the internalization of HSP27 into cells as a cell membrane receptor for HSP27 is yet to be established. Results: Purified THP-1 derived exosomes express the classical exosome biomarkers CD81 and MHC-II. Flow cytometry revealed that 18% and 23% of these exosomes are positive for HSP27 immunolabeling with 10 and 40 μg/ml anti-Hsp27-FITC, respectively. The fraction of positive exosomes dropped to 3% when 40 μg/ml of recombinant HSP27 (rHSP27) was co-mixed with these samples and 10 μg/ml anti-HSP27-FITC, thereby confirming immunolabeling specificity. In contrast, a FITC labeled truncated form of the C-terminus of HSP27 (referred to as rC1-FITC) did not bind to exosomes. Indeed, exogenous rHSP27 bound to exosomes potentiated a ~50% increase of triggered NF-κB signalling (p&lt;0.001) in THP-1 MΦ containing an alkaline phosphatase NF-kB reporter construct, while the truncated form of HSP27 (or rC1) did not (p=0.56). In both THP-1 MΦ and HAEC internalization of rHSP27-FITC into cells improved dramatically after exosome binding (e.g., THP-1: 157% increase, p&lt;0.0001), while rC1-FITC had no effect. Conclusion: Exosomes contain HSP27 cargo that provide a novel inter-cellular signalling mechanism for the athero-protective effects of HSP27 by activating NF-kB and facilitating HSP27 internalization into cells.

Unphosphorylated heat shock protein 27 suppresses fibroblast growth factor-2-stimulated vascular endothelial growth factor release in osteoblasts

Heat shock protein 27 (HSP27) also known as heat shock protein β1 (HSPB1) is a member of the family of small heat shock proteins ubiquitously expressed in all tissues. It has previously been demonstrated that HSP27 regulated the synthesis of osteocalcin and interleukin‑6 in osteoblast‑like MC3T3‑E1 cells. In the present study, the effect of HSP27 on basic fibroblast growth factor (FGF‑2)‑stimulated vascular endothelial growth factor (VEGF) synthesis in MC3T3‑E1 cells, was observed. The levels of VEGF release stimulated by FGF‑2 in the HSP27‑overexpressing MC3T3‑E1 cells were significantly lower compared with those in the control cells. In addition, the levels of VEGF release stimulated by FGF-2 in the phosphomimic HSP27-overexpressing cells were significantly higher compared with those in the non‑phosphorylatable HSP27‑overexpressing cells. Furthermore, no significant differences were observed in the FGF‑2‑induced phosphorylation levels of p44/p42 mitogen‑activated protein (MAP) kinase, p38 MAP kinase, stress‑activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) or p70 S6 kinase among the four types of transfected cells. These results suggested that unphosphorylated HSP27 attenuated the FGF‑2‑stimulated VEGF synthesis in osteoblasts.

Effect of the heat shock protein HSP27 on androgen receptor expression and function in prostate cancer cells

Heat shock proteins (HSP) are involved in processes of folding, activation, trafficking and transcriptional activity of most steroid receptors including the androgen receptor (AR). Accumulating evidence links rising heat shock protein 27 (HSP27) levels with the development of castration-resistant prostate cancer. In order to study the functional relationship between HSP27 and the AR, we modulated the expression of the small heat shock protein HSP27 in human prostate cancer (PC) cell lines. HSP27 protein concentrations in LNCaP and PC-3 cells were modulated by over-expression or silencing of HSP27. The effects of HSP27 on AR protein and mRNA levels were monitored by Western blotting and quantitative RT-PCR. Treatment for the AR-positive LNCaP with HSP27-specific siRNA resulted in a down-regulation of AR levels. This down-regulation of protein was paralleled by a decrease in AR mRNA. Most interestingly, over-expression of HSP27 in PC-3 cells led to a significant increase in AR mRNA although the cells were unable to produce functional AR protein. The observation that HSP27 is involved in the regulation of AR mRNA by a yet unknown mechanism highlights the complexity of HSP27-AR signaling network.

Proteome characterization of human articular chondrocytes leads to novel insights in the function of small heat-shock proteins in chondrocyte homeostasis

In recent years, studies have been initiated to disclose the proteome of human chondrocytes and cartilage. Despite these studies, comprehensive information of the chondrocyte proteome remains limited. This study aimed to further explore the proteome expressed by human knee chondrocytes, and to study the functional aspects of heat-shock protein 27 (HSP27), a protein related to the previously described alphaBcrystallin, in chondrocyte biology. Chondrocytes isolated from human knee articular cartilage were cultured in a three-dimensional alginate culture system. To simplify the protein mixtures, proteins extracted from chondrocyte cell lysates were fractionated based on hydrophobicity and molecular weight. Proteins were digested and the resulting peptides were separated and identified by an on-line two-dimensional (2-D) nanoliquid chromatography (nanoLC)-system coupled to a quadrupole time-of-flight (Qq-TOF) mass spectrometer. Differential expression analysis of HSP27 was performed by Western Blotting and quantitative polymerase chain reaction (QPCR). The effects of HSP27 on chondrocyte biology were explored by suppression of HSP27 expression induced by RNA interference (RNAi). In this study, we identified proteins with unknown functions together with membrane proteins, transcription factors and other low abundant proteins, which have not yet been described in chondrocytes. Based on previous knowledge on the related protein alphaBcrystallin, we selected HSP27 from the chondrocyte proteome database. Differential expression analysis revealed a decreased expression of HSP27 in Osteoarthritic (OA) chondrocytes. RNAi experiments revealed that HSP27 is involved in interleukin-1beta (IL-1beta) induced IL-6 secretion. These findings highlight that small HSPs, especially HSP27, play a prominent role in the maintenance of human articular chondrocyte homeostasis.

Regulation of HSP27 on NF-κB pathway activation may be involved in metastatic hepatocellular carcinoma cells apoptosis

BackgroundDuring the process of metastasis, cells are subjected to various apoptotic stimuli. Aberrant expression of apoptotic regulators often contribute to cell metastasis. Heat shock protein 27(HSP27) is confirmed as an apoptosis regulator, but its antiapoptotic mechanism in metastatic hepatocellular carcinoma (HCC) cells remains unclear.MethodsLevels of HSP27 protein and its phosphorylation in Hep3B, MHCC97L to MHCC97H cells with different metastatic potentials were determined by western blot analysis. MHCC97H cells were transfected with specific small interference RNA (siRNA) against HSP27. The in vitro migration and invasion potentials of cells were evaluated by Transwell assay. The apoptosis ratio of MHCC97H cells was analyzed by TUNEL staining and Flow Cytometry. Alteration of signal transduction pathway after HSP27 knockdown in MHCC97H cells was evaluated through a Human Q Series Signal Transduction in Cancer Gene Array analysis. Nuclear NF-κB contentration and endogenous IKK activity were demonstrated by ELISA assay. The association of IKKα, IKKβ, IκBα with HSP27 and the association between IKKβ and IKKα in MHCC97H cells were determined by co-immunoprecipitation assay followed by western blot analysis.ResultsHSP27 protein and its phosphorylation increased in parallel with enhanced metastatic potentials of HCC cells. siRNA-mediated HSP27 knockdown in MHCC97H significantly suppressed cells migration and invasion in vitro and induced cell apoptosis; the prominently altered signal transduction pathway was NF-κB pathway after HSP27 knockdown in MHCC97H cells. Furthermore, inhibition of HSP27 expression led to a significant decrease of nuclear NF-κB contentration and endogenous IKK activity. In addition, HSP27 was associated with IKKα, IKKβ, IκBα in three HCC cells above. ELISA assay and western blot analysis also showed a decrease of the association between IKKβ and IKKα, the association between phosphor-HSP27 and IKK complex, and an increase of total IκBα but reducing tendency of phosphor-IκBα when HSP27 expression was efficiently knocked down in MHCC97H cells.ConclusionAltogether, these findings revealed a possible effect of HSP27 on apoptosis in metastatic HCC cells, in which HSP27 may regulate NF-kB pathway activation.