Expression Of alphaB-crystallin Research Articles

Downregulation of AlphaB-crystallin in Retinal Pigment Epithelial Cells Exposed to Diabetes-related Stimuli In Vivo and In Vitro.

AlphaB-crystallin plays a pivotal role in many diseases. However, the involvement of alphaB-crystallin in retinal pigment epithelial (RPE) cells with diabetes stimuli remains unknown. The aim of this study is to examine the alterations of RPE cells and alphaB-crystallin expression in diabetic models in vivo and in vitro. Diabetic conditions in mice were induced by streptozotocin (STZ). The thickness of the RPE/choroid complex was measured by optical coherence tomography (OCT). Periodic acid-Schiff (PAS) staining was used to investigate the choriocapillaris in histological sections of murine eyeballs and oxidative stress was evaluated using immunofluorescence with anti-4-hydroxynonenal (HNE) antibody. AlphaB-crystallin expression was examined in the RPE/choroid complex using ELISA. Real-Time PCR was performed to evaluate the alphaB-crystallin expression in cultured human RPE cells with high glucose or following advanced glycation end-products (AGE) stimulation. In diabetic mice, OCT-based RPE/choroidal layers were thickened 2 months after STZ stimulation, where PAS-positive dilated choriocapillaris was noted. Immunoreactivity of 4-HNE was strongly observed in the RPE layer, from which a significant number of RPE cells was lost. Meanwhile, alphaB-crystallin expression in 2-month STZ mice was significantly lower compared to controls. In accordance with these results, in vitro data showed that the alphaB-crystallin expression was also significantly lower in RPE cells with high glucose or following AGE stimulation compared to untreated cells. In both types of diabetic models the expression of alphaB-crystallin was found to be downregulated in RPE cells and was associated with increased levels of oxidative stress.

Association of Alpha B-Crystallin Expression with Tumor Differentiation Grade in Colorectal Cancer Patients.

Alpha B-crystallin (CRYAB, HSPB5) belongs to the small heat shock protein (HSP) family and is highly expressed in various human cancers, suggesting a crucial role in tumor progression. However, few studies have examined CRYAB expression in colorectal cancer (CRC). In the present study, we investigated the relationship between CRYAB expression and the clinicopathological features of CRC samples. We comparatively analyzed CRYAB protein expression in 111 CRC tissues and normal adjacent colonic tissue, observing that it was significantly lower in CRC tissues than in corresponding non-cancerous tissues. Moreover, immunohistochemical analysis showed a significant correlation between CRYAB expression and high histological grade G3 (p = 0.033). In summary, our results point to its possible application as a prognostic biomarker in CRC patients.

Association between Alpha B-crystallin expression and prognosis in patients with solid tumors: A protocol for systematic review and meta-analysis.

Background:Alpha B-crystallin (CRYAB), as a small heat shock protein, may play critical roles in the tumorigenesis and progression of several kinds of human cancers. However, the prognostic value of CRYAB in solid malignancies remains controversial. The aim of the present study was to investigate the association between CRYAB expression and clinicopathology and prognosis of solid tumor patients.Methods:PubMed, Web of Science, EMBASE, Chinese National Knowledge Infrastructure, and WanFang databases were systematically searched to retrieve studies that investigated the prognostic value of CRYAB expression in various solid tumors. Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated to determine the strength of association between CRYAB expression and survival in patients with solid tumors. Odds ratios (ORs) with 95% CIs were pooled to assess the correlation between CRYAB expression and clinicopathological characteristics of patients with solid tumors.Results:A total of 17 studies, including 18 cohorts with 6000 patients, were included in this meta-analysis. Our results showed that increased CRYAB expression could predict poor overall survival (HR = 1.81, 95% CI: 1.50–2.19, P < .001), disease-free survival (HR = 1.47, 95% CI: 1.16–1.86, P = .001), and disease-specific survival (HR = 1.40, 95% CI: 1.19–1.63, P < .001) in patients with cancer. Furthermore, the high expression level of CRYAB was associated with certain phenotypes of tumor aggressiveness, such as lymph node metastasis (OR = 2.46, 95% CI: 1.48–4.11, P = .001), distant metastasis (OR = 3.34, 95% CI: 1.96–5.70, P < .001), advanced clinical stage (OR = 2.24, 95% CI: 1.24–4.08, P = .008), low OS rate (OR = 4.81, 95% CI: 2.82–8.19, P < .001), and high recurrence rate (OR = 1.38, 95% CI: 1.11–1.72, P = .004).Conclusions:CRYAB may serve as a valuable prognostic biomarker and therapeutic target in human solid tumors.

Matrine protects oligodendrocytes by inhibiting their apoptosis and enhancing mitochondrial autophagy

Stressed oligodendrocytes (OLGs) activate microglia to produce an inflammatory response, and the impairment of mitochondria further aggravates OLG damage, which is the earliest pathological change in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. Matrine (MAT), a tetracyclic quinolizine alkaloid derived from the herb radix sophorae flavescentis, has been shown to effectively ameliorate clinical signs of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, the mechanisms underlying the effect of MAT treatment need to be further studied. In the present study, we show that MAT effectively suppressed ongoing EAE, and significantly reduced the expression of caspase-3 and alpha B-crystallin in OLGs, therefore lessen OLG apoptosis, microglial activation and inflammatory factors secretion. MAT treatment also reduced the content of cytochrome c and malondialdehyde, an oxidative stress marker, in the central nervous system. In contrast, the levels of autophagy-related proteins Beclin1, microtubule-associated protein l light chain 3 and glutathione peroxidase was upregulated, hence enhancing mitochondrial autophagy and alleviating the imbalance of the oxidation/antioxidation system caused by mitochondrial damage. Our research indicates that MAT is effective in treating EAE, at least in part, by protecting OLGs through inhibiting their apoptosis and enhancing mitochondrial autophagy.

Downregulation of heat shock factor 4 transcription activity via MAPKinase phosphorylation at Serine 299.

Dysfunction of HSF4 is associated with congenital cataracts. HSF4 transcription activity is turned on and regulated by phosphorylation during early postnatal lens development. Our previous data suggested that mutation HSF4b/S299A can upregulate HSF4 transcription activity in vitro, but the biological significance of posttranslational modification on HSF4/S299 during lens development remains unclear. Here, we found that the mutation HSF4/S299A can upregulate the expression of HSP25 and alpha B-crystallin at both protein and mRNA levels in mouse the lens epithelial cell line, but HSF4/S299D does not. Using the rabbit polyclonal antibody against phospho-S299 of HSF4, we found that EGF and ectopic expression of MEK1 can increase the phosphorylation of HSF4/S299 and induce HSF4 sumoylation, and these effects are inhibited by U0126. ERK1/2 can phosphorylate the S299 in HSF4/wt but not in HSF4/S299A in the in vitro kinase assay. Functionally, ectopic MEK1 can inhibit HSF4-controled alpha B-crystallin expression but has less effect on HSF4/S299A. EGF can upregulate phospho-HSF4/S299 and downregulate alpha B-crystallin expression in P3 mouse lens, and this downregulation is suppressed by U0126. During mouse lens development, phosphorylation of HSF4/S299 is downregulated in P3 lens and upregulated in P7 and P14 lens. However, in 2 months old lens, both phosphorylation of HSF4/S299 and total HSF4 protein are decreased. Interestingly, ERK1/2 activity is lower in P3 lens than in P7 and P14 lens, which is in line with the phosphorylation of HSF4/S299. Taken together, our data demonstrate that HSF4/299 is a phosphorylation target of MEK1-ERK1/2, and phosphorylation of S299 is responsible for tuning down HSF4 transcription activity during postnatal lens development.

Heat shock factor 4 regulates the expression of HSP25 and alpha B-crystallin by associating with DEXD/H-box RNA helicase UAP56.

Heat shock factor 4 controls the transcription of small heat shock proteins (e.g., HSP25, alpha B-cyrstallin, and r-crystallin), that play important roles in modulating lens proteostasis. However, the molecular mechanism underlying HSF4-mediated transcription is still unclear. Using yeast two hybrid, we found that HSF4 interacts with the ATP-dependent DEXD/H-box RNA helicase UAP56, and their interaction in lens epithelial cell line was further confirmed by GST-pull down assay. UAP56 is a vital regulator of pre-mRNA splicing and mature mRNA nuclear export. The immunofluorescence assay showed that HSF4 and UBA56 co-localize with each other in the nucleus of lens epithelial cells. Ectopic UAP56 upregulated HSF4-controlled HSP25 and alpha B-crystallin proteins expression, while knocking down UAP56 by shRNA reversed it. Moreover, UAP56 interacts with and facilitates the nuclear exportation of HSP25 and alpha B-crystallin mRNA without impacting their total mRNA expression level. In lens tissues, both UAP56 and HSF4 are expressed in the same nucleus of lens fiber cells, and their expression levels are simultaneously reduced with fiber cell maturation. Taken together, these data suggested that UAP56 is a novel regulator of HSF4 and might upregulate HSF4's downstream mRNA maturation and nuclear exportation.

AlphaB-crystallin expression correlates with aging deficits in the peripheral nervous system

In an effort to identify factors that contribute to age-related deficits in the undamaged and injured peripheral nervous system (PNS), we noted that Brady and colleagues found that mice null for a small heat shock protein called alphaB-crystallin (αBC) developed abnormalities early in life that are reminiscent of aging pathologies. Because of our observation that αBC protein levels markedly reduce as wild-type mice age, we investigated whether the crystallin plays a role in modulating age-related deficits in the uninjured and damaged PNS. We show here that the presence of αBC correlates with maintenance of myelin sheath thickness, reducing macrophage presence, sustaining lipid metabolism, and promoting remyelination following peripheral nerve injury in an age-dependent manner. More specifically, animals null for αBC displayed a higher frequency of thinly myelinated axons, enhanced presence of Iba1+ macrophages, and fewer immunoreactive profiles of the cholesterol biosynthesis enzyme, squalene monooxygenase, before and after sciatic nerve crush injury. These findings thus suggest that αBC plays a protective and beneficial role in the aging PNS.

Heat shock factor 4 regulates lens epithelial cell homeostasis by working with lysosome and anti-apoptosis pathways

Activation of Heat shock factor 4-mediated heat shock response is closely associated with postnatal lens development. HSF4 controls the expression of small heat shock proteins (e.g. HSP25 and CRYAB) in lens epithelial cells. However, their roles in modulating lens epithelium homeostasis remain unclear. In this paper, we find that HSF4 is developmentally expressed in mouse lens epithelium and fiber tissue. HSF4 and alpha B-crystallin can selectively protect lens epithelial cells from cisplatin and H2O2 induced apoptosis by stabilizing mitochondrial membrane potential (ΔYm) and reducing ROS production. In addition, to our surprise, HSF4 is involved in upregulating lysosome activity. We found mLEC/HA-Hsf4 cells to have increased DLAD expression, lysosome acidity, cathepsin B activity, and degradation of plasmid DNA and GFP-LC3 protein when compared to mLEC/Hsf4-/- cells. Knocking down Cryab from mLEC/HA-Hsf4 cells inhibits HSF4-mediated lysosome acidification, while overexpression of CRYAB can upregulate cathepsin B activity in mLEC/Hsf4-/- cells. CRAYAB can interact with ATP6V1/A the A subunit of the H+ pump vacuolar ATPase, and is colocalized to lamp1 and lamp2 in the lysosome. Collectively, these results suggest that in addition to modulating anti-apoptosis, HSF4 is able to regulate lysosome activity by at least controlling alpha B-crystallin expression, shedding light on a novel molecular mechanism of HSF4 in regulating lens epithelial cell homeostasis.

AlphaB-crystallin expression in eutopic and ectopic endometrium among the patients with peritoneal form endometriosis: pregnancy outcome

AlphaB-crystallin expression in eutopic and ectopic endometrium among the patients with peritoneal form endometriosis: pregnancy outcome

Discovery of novel protein biomarkers in early silicosis by proteomics and identification of alpha B-crystallin

To establish 2-dimensional gel electrophoresis (2-DE) image for the early lung injury rats induced by silica dioxide and to identify differentially expressed protein biomarkers during the early stage of silicosis. The animal models of silicosis were established and morphology changes were observed by HE staining, and then the key protein biomarkers in silicosis were identified by 2-DE and matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI-TOF-MS) and verified by Western blot. The well qualified 2-DE gel images for experimental and control lung tissues were successfully established, and 40 different protein spots was observed when comparing the gel images between the experimental and control groups. Twenty of them were analyzed by MALDI-TOF-MS. A total of 13 altered proteins were identified, including alpha B-crystallin, mast cell protease 6, annexin 1, etc. The expression of alpha B-crystallin in lung was further verified by Western blot. The protein expression of alpha B-crystallin was increased significantly, suggesting that it might play an important role in the progress of silicosis.

Glucose regulates heat shock factor 1 transcription activity via mTOR pathway in HCC cell lines.

HSF1-mediated heat shock response is activated in most tumors and plays important roles in regulating tumor homeostasis. However, the signals underlying HSF1 activation is still not completely understood. In this paper, we find that glucose, the dominant tumor energy supplement, participates in regulating HSF1's activation in HCC cell lines. The immunoblotting results indicate that the phosphorylation of HSF1/S326, a hallmark of HSF1 activation, varies between the HCC cell lines (e.g., SMMC7721, HapG2, plc/prf5, and Chang-liver). Glucose, but not 2D-glucose, can induce the phosphorylation of HSF1 at S326 and upregulate the expression of HSF1's downstream alpha B-crystallin and Hsp70 as well as the none-heat shock proteins CSK2 and RBM23 in two tested hepatocellular carcinoma cell lines (prl/prf5 and SMMC7721). Rapamycin, an inhibitor of mTOR, can suppress the glucose-induced phosphorylation of HSF1/S326 and the expression of alpha B-crystallin. Knockdown of HSF1 with shRNA enhances the glucose-depletion-mediated inhibition of plc/prf5 cell proliferation. Our data reveal that HSF1 can be activated by glucose-mTOR pathway, providing an alternative pathway for targeting HSF1 in tumor therapy.

Upregulation of alphaB-crystallin expression in the substantia nigra of patients with Parkinson's disease

Parkinson's disease (PD) is one of the most devastating neurodegenerative disorders. The underlying mechanisms of the characteristic neurodegeneration in the substantia nigra (SN) are still not fully understood. To better understand the molecular events occurring in the SN of PD brain, we used the culture-derived isotope tag–based quantitative proteomics to compare the protein expression profiles in the nigral tissue of PD patients and control subjects. We identified a total of 11 differentially expressed proteins, including alphaB-crystallin (Cryab). Both the levels and pattern of Cryab expression in the SN were validated. It was revealed that Cryab was markedly upregulated in the SN of PD brain. Cryab expression was also upregulated in reactive astrocytes and microglia in a neurotoxin-induced mouse PD model. Moreover, we showed increased expression of Cryab in cytoplasmic inclusions in a subset of glial cells in Parkinsonian brain. Thus, we identified Cryab that is highly expressed in the SN of PD brain and may be involved in the glial pathology during dopaminergic neuron degeneration in PD.

G.O.3: Severe congenital actin related myofibrillar myopathy

Mutations in ACTA1 have been associated with nemaline myopathy, intranuclear rod myopathy, actin myopathy, cap myopathy, and congenital fiber type disproportion. Myofibrillar myopathies (MFM) are morphologically distinct but genetically heterogeneous muscular dystrophies arising from mutations in Z-disk related proteins. A 26-month-old boy was hypotonic and weak at birth, had significantly delayed motor development, and was mechanically ventilated and tube-fed since birth. At age 26 months he had cup-shaped low-set ears, a high-arched palate, contractures of the metacarpophalangeal and proximal interphalangeal joints, and facial diplegia with diffuse muscle weakness and reduced tendon reflexes, but had good head control and could sit with support. The EMG showed prominent fibrillation potentials and myopathic motor unit potentials. The muscle biopsy revealed abnormal variation in fiber size, fiber splitting, increased internal nuclei, vacuolar change, myofibrillar disorganization, hyaline structures, focal decreases of oxidative enzyme activity as well as scattered necrotic and regenerating fibers with marked increase of endomysial and perimysial connective tissue. Many structurally abnormal fibers displayed ectopic or abnormal expression of desmin, alphaB-crystallin, myotilin, dystrophin, and NCAM. Whole exome sequencing demonstrated a previously published in-frame insertion of two amino acids in ACTA1 . The mutant actin was expressed at ∼11% of wild-type in COS7 and C2 cells. C2 cells expressing mutant actin displayed cytoplasmic actin aggregates of different sizes whereas cells expressing wild-type actin displayed mostly filamentous actin. We conclude that (1) mutations in ACTA1 can cause MFM pathology; (2) MFM can be present at birth; (3) mutations in ACTA1 should be considered in patients with severe congenital hypotonia associated with muscle weakness and MFM pathology.

Alpha B-crystallin – a validated prognostic factor for poor prognosis in squamous cell carcinoma of the oral cavity

Conclusion: Alpha B-crystallin was found to be an independent prognostic marker for poor prognosis in oral cavity tumours. For oropharyngeal cancer, alpha B-crystallin had no prognostic value. Objective: The aim of this study was to see if earlier findings of alpha B-crystallin as an independent prognostic marker, and SPARC/osteonectin, PAI-1 and uPA as a prognostic combination for poor outcome in squamous cell carcinoma (SCC) of the head and neck could be confirmed in a new set of tumours. Methods: In a consecutive series of patients, assessed and primarily treated at a tertiary referral centre, histological sections from 55 patients with oral and SCC (OOPHSSC) with complete clinical data and follow-up were obtained. Oral and oropharyngeal tumours were studied separately. Immunohistochemical detection of alpha B-crystallin, SPARC/osteonectin, PAI-1 and uPA expression was performed. Results: Thirty-five patients had an oral tumour and 20 patients an oropharyngeal tumour. Twenty-five oral tumours stained negatively and 10 positively for alpha B-crystallin. For oropharyngeal tumours the figures were 15 negatively and 5 positively. Median disease-specific survival (DSS) for both sites was 33.8 and 11.9 months, for negative and positive alpha B-crystallin staining, respectively (p = 0.046). For the oral cavity, median DSS was 27.3 months for negative tumours and 7.5 months for positive tumours (p = 0.012). Corresponding figures for oropharyngeal tumours were 33.8 and 34.1 months (p = 0.95). Thus, significance in survival was only found in oral cavity tumours. In multivariate analyses there were no significant differences in DSS in the oropharyngeal group when adjusted for tumour size (T status) and presence of neck node metastasis (N status). In the oral cavity group, the significantly better DSS for negative tumours became even stronger when adjusted for T and N status. No statistical difference was found in DSS between positive and negative staining for SPARC/osteonectin, PAI-1 or uPA.

ALPHAB-CRYSTALLIN EXPRESSION IN EPIRETINAL MEMBRANE OF HUMAN PROLIFERATIVE DIABETIC RETINOPATHY

To examine the expression of alphaB-crystallin and its colocalization with vascular endothelial growth factor in the epiretinal membrane of human proliferative diabetic retinopathy. Ten epiretinal membranes of proliferative diabetic retinopathy and three normal retinas surgically excised were included in this study. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with alphaB-crystallin, vascular endothelial growth factor, and CD31 antibodies. AlphaB-crystallin was expressed in all epiretinal membranes examined. The immunolocalization of alphaB-crystallin was detected in the cytoplasm of CD31-positive endothelial cells, but not in normal retinal blood vessels. Furthermore, alphaB-crystallin immunoreactivity was colocalized in vascular endothelial growth factor-positive endothelial cells in proliferative diabetic retinopathy membranes. AlphaB-crystallin was expressed in proliferative diabetic retinopathy membranes, and colocalized with vascular endothelial growth factor-positive neovessels. AlphaB-crystallin may play a potential role in the pathogenesis of epiretinal membranes in proliferative diabetic retinopathy, together with vascular endothelial growth factor.

AlphaB-crystallin (aBC) expression in breast cancer brain metastases (BM) and primary breast cancer (pBC) with eventual BM and association with outcome.

2041 Background: aBC, a small heat shock protein, is commonly expressed in basal BC and predicts poor survival in BC patients (pts). aBC has been associated with the development of BM within basal ...

Infantile muscular dystrophy in Canadian aboriginals is an αB‐crystallinopathy

A recessively transmitted fatal hypertonic infantile muscular dystrophy has been described in Canadian aboriginals. The affected infants present with progressive limb and axial muscle stiffness and develop severe respiratory insufficiency, and most die in the first year of life. We sought to determine the genetic basis of this disease. We performed histochemical, immunocytochemical, electron microscopy, and molecular genetic studies in a cohort of 12 patients affected by this disease. Conventional histochemical and electron microscopy studies suggested myofibrillar myopathy (MFM). Therefore, we searched for ectopic expression of multiple proteins typical of MFM. Alpha B-crystallin (αBC) expression was absent from all fibers using a monoclonal antibody raised against the entire protein. However, a monoclonal antibody directed against the first 10 residues of αBC immunostained portions of abnormal fibers. Pursuing this clue, we searched for mutations in the gene for αBC (CRYAB) in available DNA samples of 8 patients. All harbored a homozygous deletion, c.60C, predicting a Ser to Ala change at codon 21 and a stop codon after 23 missense residues (p.Ser21AlafsX24). Clinically unaffected parents were heterozygous for this mutation. The homozygous c.60delC in CRYAB pinpoints the genetic basis of the fatal infantile hypertonic muscular dystrophy of Canadian aboriginals. MFMs are typically transmitted by dominant inheritance, but in this disease the parental phenotype is rescued by limited expression of the highly truncated nonfunctional mutant gene product. The severe patient phenotype is due to homozygosity for the markedly hypomorphic allele. Ann Neurol, 2011.

231 THE SMALL HEAT-SHOCK PROTEIN HSP27 SHOWS DECREASED EXPRESSION IN OA-CHONDROCYTES AND MEDIATES IL-6 SECRETION IN HUMAN ARTICULAR CHONDROCYTES

Background: HSP27 (HSPb1) is one of the small heat-shock proteins (sHSP), a protein family characterized by a molecular weight below 30 kDa. Recently, we reported the differential expression of alphaBcrystallin, another sHSP (Lambrecht et al, Arthritis Rheum, 2009). Based on the functional and structural relationship between alphaBcrystallin and HSP27, we further performed differential expression analysis on HSP27 Objectives: We aimed to achieve further insights in the involvement of small heat-shock proteins, more specific HSP27, in the biology of chondrocytes. Methods: Western blot and real-time RT-PCR analysis were performed to determine the expression levels of HSP27 in healthy and OA chondrocytes cultured in alginate beads. RNA-interference mediated gene knock-down was used to explore the role of this small heat shock protein in IL-1b activated pathways by transfecting low concentrations of siRNA in cultured chondrocytes. Upon knock-down of HSP27, cells were stimulated by IL-1b and IL-6 concentrations were determined by ELISA. Results: Western blot of healthy and OA chondrocyte lysates showed a decreased abundance of HSP27 in OA. Moreover, real-time RT-PCR confirmed the differential expression at the mRNA-level between chondrocytes isolated from visually intact and visually damaged zones of OA cartilage. The pro-inflammatory cytokines IL-1beta and TNF-alpha, both down regulated HP27 expression. Transfection of low concentrations siRNA in cultured chondrocytes resulted in an efficient knock down of HSP27 gene expression. This decreased HSP27 expression results in a reduced secretion of IL-6, in response to IL-1b. Conclusion: This study adds to the evidence that small heat-shock proteins may be important mediators in chondrocyte biology during the development of OA. Our study showed the involvement of HSP27 in IL-1b induced IL-6 secretion in human articular chondrocytes.

Role of Phospholipase D2/Phosphatidic Acid Signal Transduction in μ- and δ-Opioid Receptor Endocytosis

We demonstrated recently that opioid-induced activation of phospholipase D2 (PLD2) enhances mu- (MOPr) and delta-opioid receptor endocytosis/recycling and thus reduces the development of opioid receptor desensitization and tolerance. However, the mechanistic basis for the PLD2-mediated induction of opioid receptor endocytosis is currently unknown. Here we show that PLD2-generated phosphatidic acid (PA) might play a key role in facilitating the endocytosis of opioid receptors. However, PLD2-derived PA is known to be further converted to diacylglycerol (DAG) by PA phosphohydrolase (PPAP2). In fact, blocking of PA phosphohydrolase activity by propranolol or PPAP2-short interfering RNA (siRNA) transfection significantly attenuated agonist-induced opioid receptor endocytosis. The primary importance of PA-derived DAG in the induction of opioid receptor endocytosis was further supported by the finding that increasing the DAG level by inhibiting the reconversion of DAG into PA with the DAG kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) or the addition of the synthetic cell-permeable DAG analog 1,2-dioctanoyl-sn-glycerol (DOG), further increased the agonist-induced opioid receptor endocytosis. Moreover, the addition of DOG bypasses the PLD2-siRNA- or PPAP2-siRNA-mediated impairment of DAG synthesis and resulted in a restoration of agonist-induced opioid receptor internalization. Further studies established a functional link between PA-derived DAG and the activation of p38 mitogen-activated protein kinase (MAPK) and the subsequent phosphorylation of the Rab5 effector early endosome antigen 1, which has been demonstrated recently to be required for the induction of MOPr endocytosis. Taken together, our results revealed that the regulation of opioid receptor endocytosis by PLD2 involves the conversion of its product PA to DAG resulting in an activation of the p38 MAPK pathway.

An invisible power

Activation of Heat shock factor 4-mediated heat shock response is closely associated with postnatal lens development. HSF4 controls the expression of small heat shock proteins (e.g. HSP25 and CRYAB) in lens epithelial cells. However, their roles in modulating lens epithelium homeostasis remain unclear. In this paper, we find that HSF4 is developmentally expressed in mouse lens epithelium and fiber tissue. HSF4 and alpha B-crystallin can selectively protect lens epithelial cells from cisplatin and H2O2 induced apoptosis by stabilizing mitochondrial membrane potential (ΔYm) and reducing ROS production. In addition, to our surprise, HSF4 is involved in upregulating lysosome activity. We found mLEC/HA-Hsf4 cells to have increased DLAD expression, lysosome acidity, cathepsin B activity, and degradation of plasmid DNA and GFP-LC3 protein when compared to mLEC/Hsf4-/- cells. Knocking down Cryab from mLEC/HA-Hsf4 cells inhibits HSF4-mediated lysosome acidification, while overexpression of CRYAB can upregulate cathepsin B activity in mLEC/Hsf4-/- cells. CRAYAB can interact with ATP6V1/A the A subunit of the H+ pump vacuolar ATPase, and is colocalized to lamp1 and lamp2 in the lysosome. Collectively, these results suggest that in addition to modulating anti-apoptosis, HSF4 is able to regulate lysosome activity by at least controlling alpha B-crystallin expression, shedding light on a novel molecular mechanism of HSF4 in regulating lens epithelial cell homeostasis.