Prostate epithelial-specific expression of activated PI3K drives stromal collagen production and accumulation.

We genetically engineered expression of an activated form of P110 alpha, the catalytic subunit of PI3K, in mouse prostate epithelium to create a mouse model of direct PI3K activation (Pbsn-cre4Prb;PI3KGOF/+ ). We hypothesized that direct activation would cause rapid neoplasia and cancer progression. Pbsn-cre4Prb;PI3KGOF/+ mice developed widespread prostate intraepithelial hyperplasia, but stromal invasion was limited and overall progression was slower than anticipated. However, the model produced profound and progressive stromal remodeling prior to explicit epithelial neoplasia. Increased stromal cellularity and inflammatory infiltrate were evident as early as 4 months of age and progressively increased through 12 months of age, the terminal endpoint of this study. Prostatic collagen density and phosphorylated SMAD2-positive prostatic stromal cells were expansive and accumulated with age, consistent with pro-fibrotic TGF-ß pathway activation. Few reported mouse models accumulate prostate-specific collagen to the degree observed in Pbsn-cre4Prb;PI3KGOF/+ . Our results indicate a signaling process beginning with prostatic epithelial PI3K and TGF-ß signaling that drives prostatic stromal hypertrophy and collagen accumulation. These mice afford a unique opportunity to explore molecular mechanisms of prostatic collagen accumulation that is relevant to cancer progression, metastasis, inflammation and urinary dysfunction. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

JNK is antagonized to ensure the correct number of interommatidial cells pattern the Drosophila retina.

Apoptosis is crucial during the morphogenesis of most organs and tissues, and is utilized for tissues to achieve their proper size, shape and patterning. Many signaling pathways contribute to the precise regulation of apoptosis. Here we show that Jun N-terminal Kinase (JNK) activity contributes to the coordinated removal of interommatidial cells via apoptosis in the Drosophila pupal retina. This is consistent with previous findings that JNK activity promotes apoptosis in other epithelia. However, we found that JNK activity is repressed by Cindr (the CIN85 and CD2AP ortholog) in order to promote cell survival. Reducing the amount of Cindr resulted in ectopic cell death. Increased expression of the Drosophila JNK basket in the setting of reduced cindr expression was found to result in even more severe apoptosis, whilst ectopic death was found to be reduced if retinas were heterozygous for basket. Hence Cindr is required to properly restrict JNK-mediated apoptosis in the pupal eye, resulting in the correct number of interommatidial cells. A lack of precise control over developmental apoptosis can lead to improper tissue morphogenesis.

Preclinical evidence of sphingosine kinase 1 inhibition in alleviation of intestinal epithelial injury in polymicrobial sepsis.

BACKGROUND: Intestinal epithelial injury in septic patients predicts subsequent development of multiple organ failure, but its regulation by host factors remains unclear. Sphingosine kinase 1 is an enzyme-regulating inflammatory response. METHODS: Cecal ligation and puncture was used to induce sepsis in C57BL/6 mice with and without N,N-dimethylsphingosine, a SphK1 inhibitor. Symptom severity was monitored by murine sepsis severity score. The intestinal barrier function was determined using 4KDa fluorescein-dextran. Bacterial load in the bloodstream was determined by 16S rRNA gene amplification. RESULTS AND CONCLUSIONS: Our preliminary experimental data showed that expression of sphingosine kinase 1 in ileum was increased by sixfold in septic mice. Pharmacological blockade of sphingosine kinase 1 alleviated septic symptoms. The intestinal permeability and bacterial load in the bloodstream were also reduced in these animals. We hypothesized that inhibition of sphingosine kinase 1 may reduce pro-inflammatory cytokine production, and alleviate intestinal epithelial injury during sepsis. Further mechanistic studies and clinical specimen analyses are warranted.

Epithelial origin of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, allergen-driven inflammatory disease of the esophagus characterized predominantly by eosinophilic inflammation, leading to esophageal dysfunction. Converging data have placed the esophageal epithelium at the center of disease pathogenesis. In particular, the main EoE disease susceptibility loci at 2p23 and 5p22 encode for gene products that are produced by the esophageal epithelium: the intracellular protease calpain 14 and thymic stromal lymphopoietin, respectively. Furthermore, genetic and functional data establish a primary role for impaired epithelial barrier function in disease susceptibility and pathoetiology. Additionally, the EoE transcriptome, a set of genes dysregulated in the esophagi of patients with EoE, is enriched in genes that encode for proteins involved in esophageal epithelial cell differentiation. This transcriptome has a high proportion of esophagus-specific epithelial genes that are notable for the unexpected enrichment in genes encoding for proteases and protease inhibitors, as well as in IL-1 family genes, demonstrating a previously unappreciated role for innate immunity responses in the esophagus under homeostatic conditions. Among these pathways, basal production of the serine protease inhibitor, Kazal-type 7 (SPINK7) has been demonstrated to be part of the normal differentiation program of esophageal epithelium. Profound lost expression of SPINK7 occurs in patients with EoE and is sufficient for unleashing increased proteolytic activity (including urokinase plasminogen activator), impaired barrier function, and production of large quantities of proinflammatory and proallergic cytokines, including thymic stromal lymphopoietin. Collectively, we put forth a model in which the esophagus is normally equipped as an anti-inflammatory sensing organ and that defects in this pathway, mediated by epithelial protease/protease inhibitor imbalances, unleash inflammatory responses resulting in disorders, such as EoE.

Src Cooperates with Oncogenic Ras in Tumourigenesis via the JNK and PI3K Pathways in Drosophila epithelial Tissue.

The Ras oncogene (Rat Sarcoma oncogene, a small GTPase) is a key driver of human cancer, however alone it is insufficient to produce malignancy, due to the induction of cell cycle arrest or senescence. In a Drosophila melanogaster genetic screen for genes that cooperate with oncogenic Ras (bearing the RasV12 mutation, or RasACT), we identified the Drosophila Src (Sarcoma virus oncogene) family non-receptor tyrosine protein kinase genes, Src42A and Src64B, as promoting increased hyperplasia in a whole epithelial tissue context in the Drosophila eye. Moreover, overexpression of Src cooperated with RasACT in epithelial cell clones to drive neoplastic tumourigenesis. We found that Src overexpression alone activated the Jun N-terminal Kinase (JNK) signalling pathway to promote actin cytoskeletal and cell polarity defects and drive apoptosis, whereas, in cooperation with RasACT, JNK led to a loss of differentiation and an invasive phenotype. Src + RasACT cooperative tumourigenesis was dependent on JNK as well as Phosphoinositide 3-Kinase (PI3K) signalling, suggesting that targeting these pathways might provide novel therapeutic opportunities in cancers dependent on Src and Ras signalling.

Thymidine phosphorylase and angiogenesis in early stage esophageal squamous cell carcinoma.

BACKGROUND: The relationship between thymidine phosphorylase (TP) and angiogenesis at the early stage of esophageal squamous cell carcinoma has been unclear. METHODS: Using 14 samples of normal squamous epithelium, 11 samples of low-grade intraepithelial neoplasia, and 64 samples of superficial esophageal cancer, microvessel density (MVD) was estimated using immunostaining for CD34 and CD105. TP expression was also evaluated in both cancer cells and stromal monocytic cells (SMCs). We then investigated the correlation between MVD and TP expression in both cancer cells and SMCs. RESULTS: On the basis of the above parameters, MVD was significantly higher in cancerous lesions than in normal squamous epithelium. In terms of CD34 and CD105 expression, MVD showed a gradual increase from normal squamous epithelium, to low-grade intraepithelial neoplasia, and then to M1 and M2 cancer, and M3 or deeper cancer. M1 and M2 cancer showed overexpression of TP in both cancer cells and SMCs. There was no significant correlation between TP expression in cancer cells and MVD estimated from CD34 (rS = 0.16, P = 0.21) or CD105 (rS = 0.05, P = 0.68) expression. Significant correlations were found between TP expression in SMCs and CD34-related (rS = 0.46, P < 0.001) and CD105-related (rS = 0.34, P < 0.01) MVD. In M3 or deeper cancers, there were no significant correlations between TP expression in cancer cells or SMCs and venous invasion, lymphatic invasion, and lymph node metastasis. CONCLUSION: TP expression is activated in both cancer cells and stromal monocytic cells at the very early stage of ESCC progression. TP expression in SMCs, rather than in cancer cells, is significantly correlated with angiogenesis.

Immunolocalization of steroidogenic enzymes in the vaginal mucous of Galea spixii during the estrous cycle.

BACKGROUND: The synthesis of sex steroids is controlled by several enzymes such as17α-hydroxylase cytochrome P450 (P450c17) catalyzing androgen synthesis and aromatase cytochrome P450 (P450arom) catalyzing estrogen synthesis, both of which must complex with the redox partner NADPH-cytochrome P450 oxidoreductase (CPR) for activity. Previous studies have identified expression of steroidogenic enzymes in vaginal tissue, suggesting local sex steroid synthesis. The current studies investigate P450c17, P450aromatase and CPR expression in vaginal mucosa of Galea spixii (Spix cavy) by immuno-histochemical and western immunoblot analyses. METHODS: Stages of estrous cyclicity were monitored by vaginal exfoliative cytology. After euthanasia, vaginal tissues were retrieved, fixed and frozen at diestrus, proestrus, estrus and metestrus. The ovaries and testis were used as positive control tissues for immunohistochemistry. RESULTS: Data from cytological study allowed identification of different estrous cycle phases. Immunohistochemical analysis showed different sites of expression of steroidogenic enzymes along with tissue response throughout different phases of the estrous cycle. However, further studies are needed to characterize the derived hormones synthesized by, and the enzymes activities associated with, vaginal tissues. CONCLUSION: Current results not only support the expression of enzymes involved in sex steroid synthesis in the wall of the vagina, they also indicate that expression changes with the stage of the cycle, both the levels and types of cells exhibiting expression. Thus, changes in proliferation of vaginal epithelial cells and the differentiation of the mucosa may be influenced by local steroid synthesis as well as circulating androgens and estrogens.

Expressions of vaginal endothelial nitric oxide synthase and phosphodiesterase 5 in female sexual dysfunction: a pilot study.

INTRODUCTION AND HYPOTHESIS: The objective was to investigate the expression of endothelial nitric oxide synthase (eNOS) and phosphodiesterase (PDE) 5 in vaginal tissue of premenopausal women experiencing stress urinary incontinence (SUI) with and without sexual dysfunction. METHODS: Women presenting for treatment of SUI were screened using the Female Sexual Function Index (FSFI) and 10 were selected who met the criteria for female sexual dysfunction (FSD) and 10 asymptomatic controls. Vaginal tissue specimens were obtained from those premenopausal women aged ≥40 years who had had sexual activity ≥2 times every month for the last 6 months and who were scheduled to undergo surgery for SUI. FSD criteria was FSFI scores <18 and arousal domain scores <3. The control group had FSFI scores ≥26 and individual domain scores ≥4. The expressions of eNOS and PDE 5 were compared in the two groups using immunofluorescence staining and western blotting. RESULTS: The mean total FSFI scores were 30.4 ± 2.6 and 15.3 ± 2.3 in the control and FSD groups respectively. In immunofluorescence staining, eNOS and PDE5 were localized in the vaginal epithelium. In western blotting, the expressions of eNOS and PDE5 were significantly lower in the FSD group than in the control group (p = 0.003 and p = 0.038 respectively). CONCLUSIONS: eNOS and PDE5 in the vagina may play important roles in the pathophysiology of FSD.

The effects of hypoxia on active ionic transport processes in the gill epithelium of hyperregulating crab, Carcinus maneas.

Effects of hypoxia on the osmorespiratory functions of the posterior gills of the shore crab Carcinus maenas acclimated to 12ppt seawater (DSW) were studied. Short-circuit current (Isc) across the hemilamella (one epithelium layer supported by cuticle) was substantially reduced under exposure to 1.6, 2.0, or 2.5mg O2/L hypoxic saline (both sides of epithelium) and fully recovered after reoxygenation. Isc was reduced equally in the epithelium exposed to 1.6mg O2/L on both sides and when the apical side was oxygenated and the basolateral side solely exposed to hypoxia. Under 1.6mg O2/L, at the level of maximum inhibition of Isc, conductance was decreased from 40.0mScm-2 to 34.7mScm-2 and fully recovered after reoxygenation. Isc inhibition under hypoxia and reduced 86Rb+ (K+) fluxes across apically located K+ channels were caused preferentially by reversible inhibition of basolaterally located and ouabain sensitive Na+,K+-ATPase mediated electrogenic transport. Reversible inhibition of Isc is discussed as decline in active transport energy supply down regulating metabolic processes and saving energy during oxygen deprivation. In response to a 4day exposure of Carcinus to 2.0mg O2/L, hemolymph Na+ and Cl- concentration decreased, i.e. hyperosmoregulation was weakened. Variations of the oxygen concentration level and exposure time to hypoxia lead to an increase of the surface of mitochondria per epithelium area and might in part compensate for the decrease in oxygen availability under hypoxic conditions.

PKA-regulated VASP phosphorylation promotes extrusion of transformed cells from the epithelium.

At the early stages of carcinogenesis, transformation occurs in single cells within tissues. In an epithelial monolayer, such mutated cells are recognized by their normal neighbors and are often apically extruded. The apical extrusion requires cytoskeletal reorganization and changes in cell shape, but the molecular switches involved in the regulation of these processes are poorly understood. Here, using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry, we have identified proteins that are modulated in transformed cells upon their interaction with normal cells. Phosphorylation of VASP at serine 239 is specifically upregulated in Ras(V12)-transformed cells when they are surrounded by normal cells. VASP phosphorylation is required for the cell shape changes and apical extrusion of Ras-transformed cells. Furthermore, PKA is activated in Ras-transformed cells that are surrounded by normal cells, leading to VASP phosphorylation. These results indicate that the PKA-VASP pathway is a crucial regulator of tumor cell extrusion from the epithelium, and they shed light on the events occurring at the early stage of carcinogenesis.

Phenotype anchoring in zebrafish reveals a potential role for matrix metalloproteinases (MMPs) in tamoxifen's effects on skin epithelium.

The zebrafish is a powerful alternative model used to link phenotypes with molecular effects to discover drug mode of action. Using a zebrafish embryo-larval toxicity bioassay, we evaluated the effects of tamoxifen--a widely used anti-estrogen chemotherapeutic. Zebrafish exposed to ≥ 10 µM tamoxifen exhibited a unique necrotic caudal fin phenotype that was rapidly induced regardless of developmental life-stage when treatment was applied. To define tamoxifen's bioactivity resulting in this phenotype, targeted gene expression was used to evaluate 100 transcripts involved in tissue remodeling, calcium signaling, cell cycle and cell death, growth factors, angiogenesis and hypoxia. The most robustly misregulated transcripts in the tail were matrix metalloproteinases mmp9 and mmp13a, induced 127 and 1145 fold, respectively. Expression of c-fos, c-jun, and ap1s1 were also moderately elevated (3-7 fold), consistent with AP-1 activity--a transcription factor that regulates MMP expression. Immunohistochemistry confirmed high levels of induction for MMP13a in affected caudal fin skin epithelial tissue. The necrotic caudal fin phenotype was significantly attenuated or prevented by three functionally unique MMP inhibitors: EDTA (metal chelator), GM 6001 (broad MMP inhibitor), and SR 11302 (AP-1 transcription factor inhibitor), suggesting MMP-dependence. SR 11302 also inhibited induction of mmp9, mmp13a, and a putative MMP target, igfbp1a. Overall, our studies suggest that tamoxifen's effect is the result of perturbation of the MMP system in the skin leading to ectopic expression, cytotoxicity, and the necrotic caudal fin phenotype. These studies help advance our understanding of tamoxifen's non-classical mode of action and implicate a possible role for MMPs in tissues such as skin.

Nitric oxide inhibition of NaCl secretion in the opercular epithelium of seawater-acclimated killifish, Fundulus heteroclitus.

Nitric oxide (NO) modulates epithelial ion transport pathways in mammals, but this remains largely unexamined in fish. We explored the involvement of NO in controlling NaCl secretion by the opercular epithelium of seawater killifish using an Ussing chamber approach. Pharmacological agents were used to explore the mechanism(s) triggering NO action. A modified Biotin-switch technique was used to investigate S-nitrosation of proteins. Stimulation of endogenous NO production via the nitric oxide synthase (NOS) substrate l-arginine (2.0 mmol l-1), and addition of exogenous NO via the NO donor SNAP (10-6 to 10-4 mol l-1), decreased the epithelial short-circuit current (Isc). Inhibition of endogenous NO production by the NOS inhibitor l-NAME (10-4 mol l-1) increased Isc and revealed a tonic control of ion transport by NO in unstimulated opercular epithelia. The NO scavenger PTIO (10-5 mol l-1) supressed the NO-mediated decrease in Isc, and confirmed that the effect observed was elicited by release of NO. The effect of SNAP on Isc was abolished by inhibitors of the soluble guanylyl cyclase (sGC), ODQ (10-6 mol l-1) and Methylene Blue (10-4 mol l-1), revealing NO signalling via the sGC/cGMP pathway. Incubation of opercular epithelium and gill tissues with SNAP (10-4 mol l-1) led to S-nitrosation of proteins, including Na+/K+-ATPase. Blocking of NOS with l-NAME (10-6 mol l-1) or scavenging of NO with PTIO during hypotonic shock suggested an involvement of NO in the hypotonic-mediated decrease in Isc Yohimbine (10-4 mol l-1), an inhibitor of α2-adrenoceptors, did not block NO effects, suggesting that NO is not involved in the α-adrenergic control of NaCl secretion.

Alterations in glucose metabolism proteins responsible for the Warburg effect in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most frequent esophageal tumor in the world. ESCC presents late diagnosis, highly aggressive behavior and poor survival. Changes in tumor cell energy metabolism appear to have a prominent role in malignant transformation. Tumor cells consume glucose avidly and produce lactic acid, even under normoxia. Among the factors that may contribute to the stimulation of glycolysis in tumor cells, there are changes in the glycolytic pathway enzymes such as: pyruvate kinase M1 and M2 (PKM2 and PKM1), hexokinase II (HKII), glucose transporter isoform 1 (GLUT-1), and transcription factor induced by hypoxia (HIF1α), responsible for the transcription of proteins cited. The objective of this study is to evaluate the alterations of these proteins and their association with clinicopathological data in ESCC. We performed immunohistochemistry to determine HIF-1α, GLUT-1, PKM1, PKM2, HK2 and Ki67-expression in ESCC patients and controls. Also, we used RT-qPCR to evaluated mRNA expression of GLUT-1 in esophageal mucosa of individuals without cancer, but are alcohol drinkers and tobacco smokers. Our results showed the exclusively expression of GLUT-1 in tumors cells and dysplastic samples. We also observed a compartmentalization of the expression of PKM1 and PKM2 in relation to tumor cells and stroma associated to tumor areas. All of the proteins evaluated, excepted GLUT-1, were frequently detected in normal mucosa. No correlations between clinicopathological features and protein expressions were observed. GLUT-1 expression appears in initial tumor lesions and is maintained through ESCC evolution. We reported for the first time PKM1 staining in normal esophagus and ESCC, being mostly present in more differentiated cells.

Immunohistochemical expression of matrix metalloproteinases in ameloblastomas and pericoronal follicles.

BACKGROUND: Ameloblastoma (AM) is a benign odontogenic neoplasm characterized by local invasiveness and recurrence. We compared the immunohistochemical expression of matrix metalloproteinases in different clinical types of AM as well as in normal odontogenic tissue. METHODS: Thirteen cases of solid AMs, five cases of unicystic AM and eight pericoronal follicles (PF) were selected and subjected to immunohistochemical investigation for matrix metalloproteinase-1, matrix metalloproteinase-2 and matrix metalloproteinase-9 expressions. RESULTS: The expressions of MMP-1 and MMP-2 were very high in the cytoplasm of cells throughout the entire epithelium and in fibroblasts from the adjacent connective tissue. MMP-9 expression was observed in the same location although with weaker staining. The Kruskal-Wallis test showed statistically significant differences in the epithelial expressions of MMP-1 and MMP-2; there was lower expression among solid AMs when compared with unicystic AM and PF. Compared to both types of AM, higher stromal expression of MMP-9 was found in PF. CONCLUSION: MMP-1, MMP-2 and MMP-9 seem to be associated with AM tumour behaviour as well as physiological tissue remodelling within PF.

MWCNTs Induce ROS Generation, ERK Phosphorylation, and SOD-2 Expression in Human Mesothelial Cells.

Biological oxidative responses are involved in the toxicity of multiwall carbon nanotubes (MWCNTs), which may cause asbestos-like pathogenicity. Superoxide dismutase 2 (SOD-2) has been proposed as a biomarker of early responses to mesothelioma-inducing fibers. This study was conducted to investigate the alteration of SOD-2 expression in the human mesothelial cell lines Met-5A after exposure to nontoxic doses of MWCNTs and the potential signaling pathway. The parameters measured included the viability, morphological change, superoxide formation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and messenger RNA (mRNA)/protein levels of SOD-2. Our results showed that MWCNTs upregulated SOD-2 expression at both mRNA and protein level. Coincidently, both superoxide formation and ERK1/2 phosphorylation were observed in Met-5A cells exposed to MWCNTs and were diminished by pretreatment with the reactive oxidative species (ROS) scavenger, N-acetyl-l-(+)-cysteine (NAC). To further investigate the role of ROS/ERK1/2 in MWCNTs-induced SOD-2 overexpression, prior to MWCNTs exposure, cells were pretreated with the Mitogen-activated protein kinase kinase 1/2 (MEK 1/2) inhibitor (U0126) or with NAC. Both pretreatments decreased the MWCNTs-induced overexpression of SOD-2. These results suggest that upregulation of SOD-2 in Met-5A cells exposed to MWCNTs is mediated by ROS formation and ERK1/2 activation.

Src regulates cigarette smoke-induced ceramide generation via neutral sphingomyelinase 2 in the airway epithelium.

We previously demonstrated that the neutral sphingomyelinase (nSMase) 2 is the sole sphingomyelinase activated during cigarette smoke (CS)-induced oxidative stress of human airway epithelial cells, leading to ceramide generation and subsequent apoptosis of affected cells. Since then, we reported that nSMase2 is a phosphoprotein, the degree of enzymatic activity and stability of which are dictated by its degree of phosphorylation. Simultaneously, the non-receptor tyrosine kinase and proto-oncogene Src has increasingly become a target of interest in both smoking-related lung injury, such as chronic obstructive pulmonary disease, and lung cancer. Within this context, we tested and now present Src as a regulator of ceramide generation via modulation of nSMase2 phosphorylation and activity during CS-induced oxidative stress. Specifically, we provide evidence that Src activity is necessary for both CS-induced ceramide accumulation in vivo (129/Sv mice) and in vitro (human airway epithelial cells) and for nSMase2 activity during CS-induced oxidative stress. Moreover, because nSMase2 is exclusively phosphorylated on serines, we show that this occurs through Src-dependent activation of the serine/threonine kinase p38 mitogen-activated protein kinase during oxidative stress. Finally, we provide evidence that Src and p38 mitogen-activated protein kinase activities are critical for regulating nSMase2 phosphorylation. This study provides insights into a molecular target involved in smoking-related lung injury, represented here as nSMase2, and its modulation by the oncogene Src.

Cellular prostatic acid phosphatase, a PTEN-functional homologue in prostate epithelia, functions as a prostate-specific tumor suppressor.

The inactivation of tumor suppressor genes (TSGs) plays a vital role in the progression of human cancers. Nevertheless, those ubiquitous TSGs have been shown with limited roles in various stages of diverse carcinogenesis. Investigation on identifying unique TSG, especially for early stage of carcinogenesis, is imperative. As such, the search for organ-specific TSGs has emerged as a major strategy in cancer research. Prostate cancer (PCa) has the highest incidence in solid tumors in US males. Cellular prostatic acid phosphatase (cPAcP) is a prostate-specific differentiation antigen. Despite intensive studies over the past several decades on PAcP as a PCa biomarker, the role of cPAcP as a PCa-specific tumor suppressor has only recently been emerged and validated. The mechanism underlying the pivotal role of cPAcP as a prostate-specific TSG is, in part, due to its function as a protein tyrosine phosphatase (PTP) as well as a phosphoinositide phosphatase (PIP), an apparent functional homologue to phosphatase and tensin homolog (PTEN) in PCa cells. This review is focused on discussing the function of this authentic prostate-specific tumor suppressor and the mechanism behind the loss of cPAcP expression leading to prostate carcinogenesis. We review other phosphatases' roles as TSGs which regulate oncogenic PI3K signaling in PCa and discuss the functional similarity between cPAcP and PTEN in prostate carcinogenesis.

BAP1 (BRCA1-associated protein 1) is a highly specific marker for differentiating mesothelioma from reactive mesothelial proliferations.

The distinction between malignant mesothelioma and reactive mesothelial proliferation can be challenging both on histology and cytology. Recently, variants of the BRCA1-associated protein 1 (BAP1) gene resulting in nuclear protein loss were reported in hereditary and sporadic mesothelioma. Using immunohistochemistry, we evaluated the utility of BAP1 expression in the differential diagnosis between mesothelioma and other mesothelial proliferations on a large series of biopsies that included 212 mesotheliomas, 12 benign mesothelial tumors, and 42 reactive mesothelial proliferations. BAP1 stain was also performed in 70 cytological samples (45 mesotheliomas and 25 reactive mesothelial proliferations). BAP1 was expressed in all benign mesothelial tumors, whereas 139/212 (66%) mesotheliomas were BAP1 negative, especially in epithelioid/biphasic compared with sarcomatoid/desmoplastic subtypes (69% vs 15%). BAP1 loss was homogeneous in neoplastic cells except for two epithelioid mesotheliomas showing tumor heterogeneity. By fluorescence in situ hybridization, BAP1 protein loss was paralleled by homozygous deletion of the BAP1 locus in the vast majority of BAP1-negative tumors (31/41, 76%), whereas 9/10 BAP1-positive mesotheliomas were normal. In biopsies interpreted as reactive mesothelial proliferation BAP1 loss was 100% predictive of malignancy, as all 6 cases subsequently developed BAP1-negative mesothelioma, whereas only 3/36 (8%) BAP1-positive cases progressed to mesothelioma. On cytology/cell blocks, benign mesothelial cells were invariably positive for BAP1, whereas 64% of mesotheliomas showed loss of protein; all 6 cases showing BAP1 negativity were associated with histological diagnosis of BAP1-negative mesothelioma. BAP1 stain also showed utility in the differential of mesothelioma from most common pleural and peritoneal mimickers, such as lung and ovary carcinomas, with specificity and sensitivity of 99/70% and 100/70%, respectively. Our results show that BAP1 protein is frequently lost in mesothelioma, especially of epithelioid/biphasic subtype and is commonly associated with homozygous BAP1 deletion. BAP1 immunostain represents an excellent biomarker with an unprecedented specificity (100%) in the distinction between benign and malignant mesothelial proliferations. Finding BAP1 loss in mesothelial cells should prompt to immediately reevaluate the patient; moreover, it might be useful in mapping tumor extent and planning surgical resection.

Damage to lens fiber cells causes TRPV4-dependent Src family kinase activation in the epithelium.

The bulk of the lens consists of tightly packed fiber cells. Because mature lens fibers lack mitochondria and other organelles, lens homeostasis relies on a monolayer of epithelial cells at the anterior surface. The detection of various signaling pathways in lens epithelial cells suggests they respond to stimuli that influence lens function. Focusing on Src Family Kinases (SFKs) and Transient Receptor Potential Vanilloid 4 (TRPV4), we tested whether the epithelium can sense and respond to an event that occurs in fiber mass. The pig lens was subjected to localized freeze-thaw (FT) damage to fibers at posterior pole then the lens was incubated for 1-10 min in Krebs solution at 37 °C. Transient SFK activation in the epithelium was detectable at 1 min. Using a western blot approach, the ion channel TRPV4 was detected in the epithelium but was sparse or absent in fiber cells. Even though TRPV4 expression appears low at the actual site of FT damage to the fibers, SFK activation in the epithelium was suppressed in lenses subjected to FT damage then incubated with the TRPV4 antagonist HC067047 (10 µM). Na,K-ATPase activity was examined because previous studies report changes of Na,K-ATPase activity associated with SFK activation. Na,K-ATPase activity doubled in the epithelium removed from FT-damaged lenses and the response was prevented by HC067047 or the SFK inhibitor PP2 (10 µM). Similar changes were observed in response to fiber damage caused by injection of 5 µl hyperosmotic NaCl or mannitol solution beneath the surface of the posterior pole. The findings point to a TRPV4-dependent mechanism that enables the epithelial cells to detect remote damage in the fiber mass and respond within minutes by activating SFK and increasing Na,K-ATPase activity. Because TRPV4 channels are mechanosensitive, we speculate they may be stimulated by swelling of the lens structure caused by damage to the fibers. Increased Na,K-ATPase activity gives the lens greater capacity to control ion concentrations in the fiber mass and the Na,K-ATPase response may reflect the critical contribution of the epithelium to lens ion homeostasis.

Spontaneously Occurring Formation of Intranuclear and Cytoplasmic Inclusions in Renal Proximal Epithelium Due to Accumulation of D-Amino Acid Oxidase in Wistar Hannover Rats.

Intranuclear and cytoplasmic inclusions in the renal proximal tubular epithelium were observed in nontreated male and female Wistar Hannover rats in a 26-week study (32 weeks of age) and a 104-week study (110 weeks of age). The incidence rates were less than 5% in these two studies. In affected animals, the inclusions were observed in more than 60% of proximal tubular epithelium as various sized (approximately 1-8 µm in diameter) round and eosinophilic materials, but not in distal tubules, Henle's loop, or collecting ducts. Ultrastructurally, inclusions appeared finely granular, homogenous with middle-electron density, and without a limiting membrane. These inclusions were determined to be protein histochemically stained by Azan-Mallory and immunoreactive with an antibody against D-amino acid oxidase (DAO). There was no abnormality in in-life observations or in clinical test values suggestive of renal dysfunction. There were no associated degenerative or inflammatory changes in the kidneys, and no similar inclusions were observed in the other organs. These inclusions are very similar to propiverine hydrochloride (propiverine) and norepinephreine/serotonin reuptake inhibitor-induced inclusions. This is the first report of accumulation of DAO and formation of inclusions occurring spontaneously in rat kidneys. The data are important for toxicological studies using Wistar Hannover rats.