Apoptosis, autophagy and atherosclerosis: Relationships and the role of Hsp27.

Atherosclerosis is a multifactorial chronic inflammatory disease of the arterial wall, and an important pathological basis of coronary heart disease. Endothelial cells, vascular smooth muscle cells, and macrophages play important roles in the development of atherosclerosis. Of note, apoptosis and autophagy, two types of programmed cell death, influence the development and progression of atherosclerosis via the modulation of such cells. The small heat shock protein Hsp27 is a multifunctional protein induced by various stress factors and has a protective effect on cells. A large number of studies have demonstrated that Hsp27 plays an important role in regulating apoptosis. Recently, some studies have suggested that Hsp27 also participates in the autophagic process. Moreover, Hsp27 is closely related to the occurrence and development of atherosclerosis. Here, we summarize the molecular mechanisms of apoptosis and autophagy and discuss their effects on endothelial cells, vascular smooth muscle cells, and macrophages in the context of atherosclerotic procession. We further explore the involvement of Hsp27 in apoptosis, autophagy, and atherosclerosis. We speculate that Hsp27 may exert its anti-atherosclerotic role via the regulation of apoptosis and autophagy; this may provide the basis for the development of new approaches for the prevention and treatment of atherosclerosis.

Profiling of heat shock proteins 27 and 70 in adenoids of children.

PURPOSE: Heat shock protein (HSP)27 and 70 are molecular chaperones that may have immunomodulatory functions. We determined if and at what levels each are expressed in the adenoids of pediatric subjects. We also examined tissue distributions, associated clinical characteristics, and antibacterial effects. METHODS: Western blot, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry were applied to adenoidal tissues and lavage fluids obtained from children (N = 40) undergoing adenotonsillectomy. RESULTS: Via western blot and ELISA, both HSP27 and 70 were regularly detected in adenoidal tissue and in lavage fluid samples. HSP27 was highly expressed in epithelium, whereas HSP70 showed strong subepithelial positivity and bore a significant relation to adenoidal size. Assayed levels of HSP27 and 70 correlated inversely, and their addition to culture media independently increased bacterial numbers (Staphylococcus aureus). Upon the precipitation of each from adenoidal lavage fluids, bacterial counts declined. CONCLUSIONS: HSP27 and 70 are readily expressed in the adenoids of children and may be implicated in immunologic responses.

Functional role of inflammation in the surgical injury induced vascular remodeling of male albino rats.

Our present study investigates the cellular and molecular inflammatory events in male albino rat arterial injury. Male albino rats were subjected to longitudinal incision and carotid artery clamping for the duration of 45 days. Heat shock protein (HSP) 27, HSP70, HSP47 and Nuclear Factor kappa B (NF-κB) expressions were determined by qPCR and Western blot method. The morphology of vessel wall alteration was studied by the light microscopy. The expression of NF-κB was found to be increased after ten days of carotid artery injury. The qPCR and Western blot analysis showed elevation in HSP47, HSP27, and HSP70 expression, ten days following the surgical injury. The neointima-formation and the media layer discontinuity were evidenced by light microscopy. The dendritic-like cells were in close contact with the lymphocytes. Our study reports that the surgical injury induces an inflammatory response through the increased NF-κB and HSPs expression.

The small heat shock protein Hsp27: Present understanding and future prospects.

Heat shock proteins are important for maintaining protein homeostasis and cell survival. Among different classes of highly conserved Hsps, low molecular weight Hsps (sHsps) have significant place, particularly Hsp27, whose role has been demonstrated in wide range of biological processes, including development, immunity, diseases and therapy. In this review, the structure and functions of Hsp27 and related genes, their role in different cellular processes as well as in stress tolerance, is highlighted.

Quantitative proteomic analysis shows differentially expressed HSPB1 in glioblastoma as a discriminating short from long survival factor and NOVA1 as a differentiation factor between low-grade astrocytoma and oligodendroglioma.

BACKGROUND: Gliomas account for more than 60 % of all primary central nervous system neoplasms. Low-grade gliomas display a tendency to progress to more malignant phenotypes and the most frequent and malignant gliomas are glioblastomas (GBM). Another type of glioma, oligodendroglioma originates from oligodendrocytes and glial precursor cells and represents 2-5 % of gliomas. The discrimination between these two types of glioma is actually controversial, thus, a molecular distinction is necessary for better diagnosis. METHODS: iTRAQ-based quantitative proteomic analysis was performed on non-neoplastic brain tissue, on astrocytoma grade II, glioblastoma with short and long survival and oligodendrogliomas. RESULTS: We found that expression of nucleophosmin (NPM1), glucose regulated protein 78 kDa (GRP78), nucleolin (NCL) and heat shock protein 90 kDa (HSP90B1) were increased, Raf kinase inhibitor protein (RKIP/PEBP1) was decreased in glioblastoma and they were associated with a network related to tumor progression. Expression level of heat shock protein 27 (HSPB1/HSP27) discriminated glioblastoma presenting short (6 ± 4 months, n = 4) and long survival (43 ± 15 months, n = 4) (p = 0.00045). Expression level of RNA binding protein nova 1 (NOVA1) differentiated low-grade oligodendroglioma and astrocytoma grade II (p = 0.0082). Validation were done by Western blot, qRT-PCR and immunohistochemistry in a larger casuistry. CONCLUSION: Taken together, our quantitative proteomic analysis detected the molecular triad, NPM1, GRP78 and RKIP participating together with NCL and HSP27/HSPB1 in a network related to tumor progression. Additionally, two new important targets were uncovered: NOVA1 useful for diagnostic refinement differentiating astrocytoma from oligodendroglioma, and HSPB1/HSP27, as a predictive factor of poor prognosis for GBM.

A combined laser microdissection and mass spectrometry approach reveals new disease relevant proteins accumulating in aggregates of filaminopathy patients.

Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actin-binding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation.

Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

1,3,5-Trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone directly targets heat shock protein 27 in hepatocellular carcinoma.

We previously showed that the small molecule 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone (TDP) induces apoptosis in hepatocellular carcinoma (HCC) by suppressing Hsp27 expression, although the mechanism is not fully understood. To investigate the functional association between TDP and Hsp27 protein in HCC, recombinant Hsp27 protein was incubated with TDP at room temperature, and assayed by mass spectrum (MS) and natural electrophoresis. TDP effectively stimulated Hsp27 to form aggregates ex vitro, leading to suppression of its chaperone activity. The aggregates were degraded by the ubiquitin-proteasome (UPS) pathway. TDP directly interacted with Asp17 and Phe55 in chain C of Hsp27 on the basis of bioinformatic prediction. In conclusion, Hsp27 is a direct target of TDP in its anti-cancer activity, which provides strong support for a clinical application.

Cytoprotective mechanisms in rats lung parenchyma with zinc deprivation.

Suboptimal intake of Zinc (Zn) is one of the most common worldwide nutritional problems. The aim of this study is to provide new evidence on the relation between moderate Zn restriction, and cytoprotective functions in airway epithelium. We analyzed the effect of moderate Zn deficiency (ZD) on the expression of several pro and anti-apoptotic proteins and cytoprotective factors (Hsp27 and Hsp 70i), as well as the effect of restoring Zn during the refeeding period. Adult male rats were divided into three groups: Zn-adequate control group, Zn-deficient group and Zn-refed group. Our previous findings showed an important oxidative and nitrosative stress during ZD, this situation is accompanied by inflammation and alterations in the expression of matrix extracellular proteins. We observed a strong immunopositive area of anti and pro-apoptotics proteins in ZD groups. The mRNA levels of Nrf-2, Bax and Bad were increased in ZD, while in ZD refed group its levels were similar to the control values. The increased expression of Nrf-2 is likely to be critical for protection of lung under inflammatory process triggered during ZD. Hsp27 and Hsp 70i showed an increase of immunostaining area but they were not significant. During the supplementation period, heat-shock proteins increased significantly. In conclusion, our results provide further evidence of the pathways involved in cytoprotection and apoptosis caused by ZD. Additional studies are required in order to investigate whether Hsp27 and Hsp70 are consistently associated with cellular stress and inflammation in lung. There may be a beneficial role for improved Zn nutrition or Zn supplements early in lung pathology.

2,4-Bis(4-hydroxybenzyl)phenol inhibits heat shock transcription factor 1 and sensitizes lung cancer cells to conventional anticancer modalities.

Heat shock factor 1 (HSF1) is a transcription factor that regulates expression of heat shock protein (HSP) genes in response to stress. HSPs are expressed at high levels in a wide range of tumors. It has been reported that HSF1 and HSPs are associated closely in tumorigenesis. In the present study, a screen was performed using a luciferase reporter under the control of a heat shock element to find inhibitors of HSF1 activity, and 2,4-bis(4-hydroxybenzyl)phenol (1), isolated from the rhizomes of Gastrodia elata, was identified as an active compound. This substance effectively inhibited HSF1 activity and decreased levels of HSP27 and HSP70. Compound 1 induced the degradation of HSF1 protein through dephosphorylation of HSF1 on S326, which decreases HSF1 protein stability. In addition, 1 also induced growth arrest and apoptosis of NCI-H460 human lung cancer cells. Markers of apoptosis, such as cleaved PARP and cleaved caspase-3, were detected after treatment with 1. Furthermore, cotreatment with 1 and conventional anticancer modalities such as paclitaxel, cisplatin, or ionizing radiation potentiated their effects on lung cancer cells. These results suggest that inhibition of HSF1 by 1 may help overcome resistance to conventional anticancer modalities in HSF1-overexpressed cancer cells.

B7H4, HSP27 and DJ-1 molecular markers as prognostic factors in pancreatic cancer.

OBJECTIVES: Pancreatic cancer (PC) is one of the most lethal tumors of the gastrointestinal tract. The ability to predict which patients would benefit most from surgical intervention and chemotherapy would be a great clinical tool. A large number of potential markers have been identified lately in pancreatic cancer and their clinical utilities as prognostic tools are under investigation. METHODS: We recruited 41 patients who had undergone radical surgical resection for PC between 2003 and 2010. To investigate the prognostic factors, we evaluated 3 possible markers: B7H4, HSP27 and DJ-1 protein expressions in the tissue specimens of these 41 patients by immunohistochemistry and analyzed the clinical and pathological features of these specimens. RESULTS: The expression of the three antigens was independently associated with a negative impact of chemotherapy with gemcitabine on patient's survival. Moreover, patients who overexpressed B7H4 had worse prognosis than the ones who did not. CONCLUSIONS: B7H4, DJ-1 and HSP27 may be used in the future as prognostic markers that express resistance of pancreatic cancer patients to chemotherapy with gemcitabine.

Analysis of the expression of heat-shock protein 27 in patients with oral lichen planus.

OBJECTIVE: Heat-shock protein 27 (hsp27) has been implicated in several biological events. In this experimental study, we aimed at analysing, for the first time, the expression of hsp27 in the diverse stages of oral lichen planus (OLP) lesions. MATERIALS AND METHODS: Thirty-six biopsy specimens of patients with OLP and 10 of healthy patients were selected. OLP specimens were divided into three groups: G1 - moderate or mildly active OLP; G2 - active or moderately active atrophic OLP; G3 - mild or inactive atrophic OLP. Hsp27 expression was analysed by immunohistochemistry (staining intensity and percentage of stained cells), and results of staining were compared between the different groups. Gender, age and anatomical location were also studied. RESULTS: In the basal layer, an increase of hsp27 expression in both G2 and G3 was observed when compared to G1 and control group. In contrast, a decrease of hsp27 expression in the superficial layer was observed in all groups when compared to control group. CONCLUSION: The increased expression of Hsp27 in the basal layer observed during the OLP evolution and the less staining in the superficial layers in all cases of OLP suggest that hsp27 may have a role in the OLP pathogenesis.

Regenerative capability of dental pulp cells after crown fracture.

The purpose of this study was to evaluate the characteristics of dental pulp cells for tissue engineering derived from the fractured incisal portion of tooth crowns. Thirty Sprague-Dawley rats were used for histological and immunohistochemical analysis of nestin protein expression and to measure levels of mRNAs encoding osteocalcin, osteopontin, bone sialoprotein (BSP), dentin sialoprotein (DSP), heat shock protein (HSP) 27, vascular endothelial growth factor (VEGF), ATP-binding cassette transporter G2 (ABCG2), nestin, and p57(Kip2) . Odontoblasts at the incisal portion in the control group were oriented in a regular pattern, but those in the experimental group were randomly stratified. Immunohistochemically, only a few odontoblasts were positive for nestin at the incisal portion in the experimental group at 2 days. Some cells in the inner area in the control group were positive for nestin, but nestin-positive cells in the experimental group at the incisal portion were not observed. The mRNA expression for osteogenic or odontogenic markers in the experimental group was higher than in the control group. HSP27 mRNA expression in the experimental group at 2 days was higher than in the control group and in the experimental group at 7 days. mRNA expression of stem cell markers, such as ABCG2 and nestin, in the experimental group tended to decrease compared with the control. In conclusion, this study demonstrates that dental pulp stem cells derived from fractured teeth differentiate to osteogenic or odontogenic cells.

Stress protein Hsp27 expression predicts the outcome in operated small cell lung carcinoma and large cell neuroendocrine carcinoma patients.

PURPOSE: Heat shock protein (Hsp)27 is overexpressed in a range of human cancers and is implicated in tumor cell proliferation, differentiation, invasion, metastasis, and survival. The aim of the present study was to determine the prognostic significance of Hsp27 expression in small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC). METHODS: Surgically resected SCLCs (N=51) and LCNECs (N=15) were studied. The Hsp27 expression was detected immunohistochemically. RESULTS: Hsp27 positive immunoreaction in the cytoplasm was observed in 45 (88%) SCLCs and 14 (93%) LCNECs. A combination of cytoplasmic with nuclear Hsp27 expression was observed in 28 (62%) SCLCs and 14 (100%) LCNECs. There was a correlation between Hsp27 cytoplasmic overexpression and Hsp27 nuclear expression with patient survival confirmed by Cox multivariate analysis. CONCLUSION: We conclude that the higher Hsp27 cytoplasmic expression and nuclear expression may represent favorable prognostic factors in SCLC and LCNEC.

HSP27/HSPB1 as an adaptive podocyte antiapoptotic protein activated by high glucose and angiotensin II.

Apoptosis is a driving force of diabetic end-organ damage, including diabetic nephropathy (DN). However, the mechanisms that modulate diabetes-induced cell death are not fully understood. Heat shock protein 27 (HSP27/HSPB1) is a cell stress protein that regulates apoptosis in extrarenal cells and is expressed by podocytes exposed to toxins causing nephrotic syndrome. We investigated the regulation of HSPB1 expression and its function in podocytes exposed to factors contributing to DN, such as high glucose and angiotensin (Ang) II. HSPB1 expression was assessed in renal biopsies from patients with DN, minimal change disease or focal segmental glomerulosclerosis (FSGS), in a rat model of diabetes induced by streptozotocin (STZ) and in Ang II-infused rats. The regulation of HSPB1 was studied in cultured human podocytes and the function of HSPB1 expressed in response to pathophysiologically relevant stimuli was explored by short interfering RNA knockdown. Total kidney HSPB1 mRNA and protein expression was increased in rats with STZ-induced diabetes and in rats infused with Ang II. Upregulation of HSPB1 protein was confirmed in isolated diabetic glomeruli. Immunohistochemistry showed increased glomerular expression of HSPB1 in both models and localized glomerular HSPB1 to podocytes. HSPB1 protein was increased in glomerular podocytes from patients with DN or FSGS. In cultured human podocytes HSPB1 mRNA and protein expression was upregulated by high glucose concentrations and Ang II. High glucose, but not Ang II, promoted podocyte apoptosis. HSPB1 short interfering RNA (siRNA) targeting increased apoptosis in a high-glucose milieu and sensitized to Ang II or TGFß1-induced apoptosis by promoting caspase activation. In conclusion, both high glucose and Ang II contribute to HSPB1 upregulation. HSPB1 upregulation allows podocytes to better withstand an adverse high-glucose or Ang II-rich environment, such as can be found in DN.

Toxicoproteomic analysis of a mouse model of nonsteroidal anti-inflammatory drug-induced gastric ulcers.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are valuable agents; however, their use has been limited by their association with mucosal damage in the upper gastrointestinal tract. NSAIDs inhibit cyclooxygenase and consequently block the synthesis of prostaglandins, which have cytoprotective effects in gastric mucosa; these effects on prostaglandins have been thought to be major cause of NSAID-induced ulceration. However, studies indicate that additional NSAID-related mechanisms are involved in formation of gastric lesions. Here, we used a toxicoproteomic approach to understand cellular processes that are affected by NSAIDs in mouse stomach tissue during ulcer formation. We used fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS)-which consists of fluorogenic derivatization, separation and fluorescence detection by LC, and identification by LC-tandem mass spectrometry-in this proteomic analysis of pyrolic stomach from control and diclofenac (Dic)-treated mice. FD-LC-MS/MS results were highly sensitive; 10 differentially expressed proteins were identified, and all 10 were more highly expressed in Dic-treated mice than in control mice. Specifically, expression levels of 78 kDa glucose-regulated protein (GRP78), heat shock protein beta-1 (HSP27), and gastrin were more than 3-fold higher in Dic-treated mice than in control mice. This study represents a first step to ascertain the precise actors of early NSAID-induced ulceration.

Phosphorylation-dependent subcellular localization of the small heat shock proteins HspB1/Hsp25 and HspB5/αB-crystallin in cultured hippocampal neurons.

The so-called stress response involving upregulation of heat shock proteins (Hsps) is a powerful mechanism of cells to deal with harmful conditions to which they are exposed throughout life, such as hyperthermia, hypoxia or oxidative stress. To gain more information about the molecular targets by which HspB1 (Hsp25) and HspB5 (αB-crystallin) might exert their neuroprotective effect we investigated the subcellular localization of unphosphorylated and phosphorylated HspB1 and B5 in neurons by immunocytochemistry and subcellular fractionation. In cultured hippocampal neurons, the unphosphorylated forms of both Hsps were localized in the perikaryon and nucleus, whereas the phosphorylated forms were recruited into neuronal processes. pHspB1-Ser15 and -Ser 86 were found within dendrites with a punctate distribution pattern partially colocalizing with the synaptic marker vGlut-1. pHspB5-Ser19 and -Ser45 localized to axons and dendrites with a filamentous-like staining pattern, whereas pHspB5-Ser59 was found in dendrites, especially along the plasma membrane and in spines. Biochemical analysis, i.e. subcellular fractionation of rat brain with subsequent Western blotting supported these localizations. These data show that in neurons HspB1 and B5 may have various molecular interaction partners at synapses, within dendrites and axons and that this interaction is likely to be regulated by phosphorylation. Stress-induced phosphorylation of HspB1 and B5 may lead to binding of these Hsps to their targets at synapses and neuronal processes which might provide one important mechanism of how they exert their neuroprotective effect.

Carotid atherosclerotic plaques: proteomics study after a low-abundance protein enrichment step.

Atherosclerosis is one of the most important causes of cardiovascular and cerebrovascular events. Although phenotypic differentiation between stable and unstable plaques is currently possible, proteomic analysis of the atherosclerotic plaque could offer a global view of the atherosclerosis pathology. With the objective to highlight the detection of low-abundance proteins, we reduced the dynamic range of proteins by combinatorial peptide ligand library treatment of human carotid artery atherosclerotic plaques. After enrichment step, abundance of major proteins was decreased, revealing different protein profiles as assessed by both SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis comparative analyses. Identification of proteins that were contained in a spot allowed finding large differences between noncomplicated and complicated plaques from carotid atherosclerotic lesions. Novel low-abundance proteins were detected correlating very well with biological alterations related to atherosclerosis (heat shock protein 27 (HSP27) isoforms, aldehyde dehydrogenase, moesin, Protein kinase C delta-binding protein, and inter-α trypsin inhibitor family heavy chain-related protein (ITIH4)). At the same time, the differential expression of known proteins of interest such as hemoglobin ß-chain and heat shock protein 27 between noncomplicated and hemorrhagic complicated plaques was maintained after enrichment step. The detection of different isoforms of a low-abundance protein such as heat shock protein 27 species was actually improved after enrichment of tissue protein extracts. All of these findings clearly support further investigations in view to confirm the role of these proteins as possible biomarkers.

Lysosomal and cytoskeletal events in epithelial salivary tumours as assessed by imunohistochemistry for CD63 and HSP27.

Little attention has been paid to immunohistochemically assessed, lysosomal activities in salivary neoplasia. In an attempt to remedy this, the present investigation applied immunohistochemistry for CD63 antigen (a lysosomal membranous protein) and HSP27 (a molecular chaperone with roles in intracellular homeostasis) to archival paraffin-embedded surgical specimens of 101 benign and malignant, epithelial salivary tumours. Diffuse cytoplasmic CD63 immunoreactivity was seen in serous cells in acinic cell carcinoma, and mucous cells in mucoepidermoid carcinoma, pleomorphic adenoma (PA) and Warthin tumour. Apical rims or bands of CD63 immunoreactivity were also seen in simple cells lining tubular structures in PA, acinic cell carcinoma, mucoepidermoid carcinoma and polymorphous (low-grade) adenocarcinoma; and, occasionally, oncocytic luminal cells in Warthin tumour. HSP27 immunoreactivity was usually seen in non-luminal cells of PA, basal cells of oncocytic tumours, epidermoid cells of mucoepidermoid carcinoma and PA, and cells outlining aggregates or pseudolumina of adenoid cystic carcinoma. Expression of CD63 is preferentially associated with differentiated or simple luminal cell phenotypes in epithelial salivary tumours and possibly reflects autophagy of secretory granules or absorption of luminal material. Expression of HSP27 is preferentially associated with non-luminal cells and possibly reflects remodelling of the cytoskeleton.

p27 suppresses arsenite-induced Hsp27/Hsp70 expression through inhibiting JNK2/c-Jun- and HSF-1-dependent pathways.

p27 is an atypical tumor suppressor that can regulate the activity of cyclin-dependent kinases and G(0)-to-S phase transitions. More recent studies reveal that p27 may also exhibit its tumor-suppressive function through regulating many other essential cellular events. However, the molecular mechanisms underlying these anticancer effects of p27 are largely unknown. In this study, we found that depletion of p27 expression by either gene knock-out or knockdown approaches resulted in up-regulation of both Hsp27 and Hsp70 expression at mRNA- and promoter-derived transcription as well as protein levels upon arsenite exposure, indicating that p27 provides a negative signal for regulating the expression of Hsp27 and Hsp70. Consistently, arsenite-induced activation of JNK2/c-Jun and HSF-1 pathways was also markedly elevated in p27 knock-out (p27(-/-)) and knockdown (p27 shRNA) cells. Moreover, interference with the expression or function of JNK2, c-Jun, and HSF-1, but not JNK1, led to dramatic inhibition of arsenite-induced Hsp27 and Hsp70 expression. Collectively, our results demonstrate that p27 suppresses Hsp27 and Hsp70 expression at the transcriptional level specifically through JNK2/c-Jun- and HSF-1-dependent pathways upon arsenite exposure, which provides additional important molecular mechanisms for the tumor-suppressive function of p27.