Segment-Dependent Gene Expression Profiling of the Cartilaginous Fish Nephron Using Laser Microdissection for Functional Characterization of Nephron at Segment Levels.

For adaptation to a high salinity marine environment, cartilaginous fishes have evolved a ureosmotic strategy. They have a highly elaborate "four-loop nephron" in the kidney, which is considered to be important for reabsorption of urea from the glomerular filtrate to maintain a high concentration of urea in the body. However, the function and regulation, generally, of the "four-loop nephron" are still largely unknown due to the complicated configuration of the nephron and its many subdivided segments. Laser microdissection (LMD) followed by RNA-sequencing (RNA-seq) analysis is a powerful technique to obtain segment-dependent gene expression profiles. In the present study, using the kidney of cloudy catshark, Scyliorhinus torazame, we tested several formaldehyde-free and formaldehyde-based fixatives to optimize the fixation methods. Fixation by 1% neutral buffered formalin for 15 min resulted in sufficient RNA and structural integrities, which allowed LMD clipping of specific nephron segments and subsequent RNA-seq analysis. RNA-seq from the LMD samples of the second-loop, the fourth-loop, and the five tubular segments in the bundle zone revealed a number of specific membrane transporter genes that can characterize each segment. Among them, we examined expressions of the Na + -coupled cotransporters abundantly expressed in the second loop samples. Although the proximal II segment of the second loop is known for the elimination of excess solutes, the present results imply that the PII segment is also crucial for reabsorption of valuable solutes. Looking ahead to future studies, the segment-dependent gene expression profiling will be a powerful technique for unraveling the renal mechanisms and regulation in euryhaline elasmobranchs.

Cholesterol Is a Regulator of CAV1 Localization and Cell Migration in Oral Squamous Cell Carcinoma.

Cholesterol plays an important role in cancer progression, as it is utilized in membrane biogenesis and cell signaling. Cholesterol-lowering drugs have exhibited tumor-suppressive effects in oral squamous cell carcinoma (OSCC), suggesting that cholesterol is also essential in OSCC pathogenesis. However, the direct effects of cholesterol on OSCC cells remain unclear. Here, we investigated the role of cholesterol in OSCC with respect to caveolin-1 (CAV1), a cholesterol-binding protein involved in intracellular cholesterol transport. Cholesterol levels in OSCC cell lines were depleted using methyl-ß-cyclodextrin and increased using the methyl-ß-cyclodextrin-cholesterol complex. Functional analysis was performed using timelapse imaging, and CAV1 expression in cholesterol-manipulated cells was investigated using immunofluorescence and immunoblotting assays. CAV1 immunohistochemistry was performed on surgical OSCC samples. We observed that cholesterol addition induced polarized cell morphology, along with CAV1 localization at the trailing edge, and promoted cell migration. Moreover, CAV1 was upregulated in the lipid rafts and formed aggregates in the plasma membrane in cholesterol-added cells. High membranous CAV1 expression in tissue specimens was associated with OSCC recurrence. Therefore, cholesterol promotes the migration of OSCC cells by regulating cell polarity and CAV1 localization to the lipid raft. Furthermore, membranous CAV1 expression is a potential prognostic marker for OSCC patients.

Identification and characterization of R2TP in the development of oral squamous cell carcinoma.

R2TP is a well-conserved molecular chaperone complex, composed of Pontin, Reptin, RPAP3, and PIH1D, in eukaryotes. Recent studies have suggested an involvement of R2TP in cancer development. However, it remains unclear if it is related to the development of oral squamous cell carcinoma (OSCC), which is the most common type of oral cancer. Here, we identify and investigate the function of R2TP in OSCC development. Immunohistochemical analysis reveals that all of the R2TP components are strongly expressed in normal oral epithelia and OSCC tissues, where actively proliferating cells are abundant. Co-immunoprecipitation assay identifies that R2TP components form a protein complex in OSCC-derived HSC4-cells. Knockdown experiments show that all R2TP components, except for RPAP3, are required for the cell proliferation and migration of HSC-4 cells. Furthermore, we reveal that Pontin contributes to a gain-of-function (GOF) activity of mutp53-R248Q in HSC-4 cells by regulating phosphorylation levels of mutp53 at Ser15 and Ser46. To our knowledge, this study is the first to report the functional involvement of R2TP and its components in the malignant characteristics of OSCC cells.

Rac1-dependent phagocytosis of apoptotic cells by oral squamous cell carcinoma cells: A possible driving force for tumor progression.

Apoptotic cell death frequently occurs in human cancer tissues including oral squamous cell carcinoma (SCC), wherein apoptotic tumor cells are phagocytosed not only by macrophages but also by neighboring tumor cells. We previously reported that the engulfment of apoptotic SCC cells by neighboring SCC cells frequently occurs at the invading front. Therefore, we hypothesized that the phagocytosis of these apoptotic cells by tumor cells contributes to disease progression. Herein, using cultured oral SCC cells, we aimed to confirm whether tumor cells actually phagocytose apoptotic cells and to examine whether cellular activities are regulated by the phagocytosis of apoptotic cells. Co-culture experiments showed that living cells could ingest apoptotic cells into phagolysosomes. NSC23766, an inhibitor of Rac1, which is a key regulator of phagocytic cup formation in professional phagocytes, dramatically suppressed the phagocytosis of apoptotic cells by living cells. Additionally, cell migration and the secretion of DKK1, a tumor-promoting protein, were enhanced by co-culture with apoptotic cells, whereas NSC23766 inhibited these effects. These results show that tumor cells can actively phagocytose apoptotic neighbors in a Rac1-dependent manner and that such activity increases their migration. The regulation of apoptotic cell phagocytosis thus represents new directions for therapeutic intervention for oral cancer.

Lower Prolactin Levels in Patients Treated With Aripiprazole Regardless of Antipsychotic Monopharmacy or Polypharmacy.

BACKGROUND: Hyperprolactinemia is a troublesome adverse effect of antipsychotics. Aripiprazole (ARP), which is one of second-generation antipsychotics, has been reported to lower serum prolactin (PRL) levels. However, few studies have compared the effect of ARP on plasma PRL levels between monopharmacy and polypharmacy with antipsychotics. METHODS: We conducted a large-scale investigation of the physical risk for inpatients with schizophrenia using a questionnaire covering demographic data, the number, dose and type of antipsychotics, and serum PRL levels. RESULTS: Sufficient data to conduct an assessment of the effect on PRL levels between antipsychotic monopharmacy and polypharmacy were obtained from 316 of the inpatients. Serum PRL levels in ARP combination group were lower than non-ARP combination group, regardless of antipsychotic monopharmacy or polypharmacy. CONCLUSIONS: The present study suggests that ARP lowers serum PRL levels regardless of monopharamacy or polypharmacy with antipsychotics.

CD36 expression on oral squamous cell carcinoma cells correlates with enhanced proliferation and migratory activity.

OBJECTIVE: Recent studies have demonstrated the pro-tumour role of CD36 in multiple cancer types. However, its role has not been well elucidated in oral squamous cell carcinoma (OSCC). Here, we aimed to evaluate the role of CD36 in proliferation and migration of OSCC cells. METHODS: Human OSCC cell lines HSC-2, HSC-3, HSC-4 and Ca9-22 were assessed for proliferation by staining with the cell proliferation marker Ki-67. We also assessed migration activity, and the expression of cell adhesion molecules such as E-cadherin and ß-catenin and platelet-derived growth factor receptors (PDGFRs) of CD36-positive cells. RESULTS: CD36-positive cells showed increased expression of Ki-67 and migration activity compared with CD36-negative cells. Moreover, CD36-positive cells showed reduced expression of E-cadherin and ß-catenin, whereas the expression of PDGFRs increased compared with that in CD36-negative cells. CONCLUSIONS: Our results strongly suggest that CD36 has an important role in facilitating the proliferation and migration activity of OSCC cells, indicating its usefulness in the diagnosis of high-grade tumour and targeted therapy of oral cancer.

High-density lipoprotein-cholesterol and antipsychotic medication in overweight inpatients with schizophrenia: post-hoc analysis of a Japanese nationwide survey.

BACKGROUND: Patients with schizophrenia have an increased prevalence of metabolic disturbances compared with the general population. However, the mechanisms underlying the metabolic side effects of antipsychotics are unknown. The aim of the present study was to compare the levels of high-density lipoprotein (HDL)-cholesterol in Japanese schizophrenia patients medicated with olanzapine, risperidone, or aripiprazole monotherapy. METHODS: This study was a post-hoc analysis of a nationwide survey, which included 433 Japanese outpatients with schizophrenia and 674 inpatients. A brief questionnaire was compiled that covered demographic data, systolic blood pressure, diastolic blood pressure, and HDL-cholesterol after reviewing the relevant literature and guidelines. To compare demographic and clinical characteristics, analysis of variance was performed for continuous variables and the chi-square test was performed for categorical variables. For comparisons of HDL-cholesterol levels among the three antipsychotic groups, analysis of covariance was carried out with age, diastolic blood pressure, chlorpromazine-equivalent dosage, and waist circumference as confounding variables after stratification by body mass index (BMI) for each outpatient group and inpatient group. RESULTS: The mean age was 57.9 ± 14.0 years and the mean BMI was 23.4 ± 4.5 kg/m2. HDL-cholesterol levels when stratified by BMI differed significantly (p = 0.019) between the three antipsychotic groups after age, diastolic blood pressure, chlorpromazine-equivalent dosage, and waist circumference in inpatients. A significant difference in HDL-cholesterol levels was only found in the overweight inpatient group, and no significant differences in HDL-cholesterol levels were found among the three antipsychotics for outpatients of all BMI stratifications or inpatients that were underweight or of normal weight. For post-hoc analysis of HDL-cholesterol levels in overweight inpatients, HDL-cholesterol was significantly lower in the olanzapine group than in the aripiprazole group (p = 0.023). CONCLUSIONS: This study reveals a difference in HDL-cholesterol levels in overweight Japanese inpatients with schizophrenia resulting from the use of different antipsychotics. In the post-hoc analysis of HDL-cholesterol levels in overweight inpatients, HDL-cholesterol was significantly lower in the olanzapine group than in the aripiprazole group. Further studies incorporating more detailed evaluations, including diet and physical activity, are needed to clarify the differences in HDL-cholesterol according to antipsychotic use.

Proteomic and histopathological characterization of the interface between oral squamous cell carcinoma invasion fronts and non-cancerous epithelia.

Oral squamous cell carcinomas (SCCs) are frequently associated with pre-invasive lesions including carcinoma in-situ (CIS), and CISs further form lateral interfaces against surrounding normal or dysplastic epithelia (ND). At the interface where keratin (K) 17 positive (+) SCC/CIS cells are in contact with K13+ ND cells, "cell competition" must be evoked between two such different cell types. Thus, the aim of this study was to characterize the histopathology of the SCC/CIS-ND interface and to determine protein profiles around the interface by proteomics. A total of 112 lateral interfaces were collected from 55 CIS and 57 SCC foci, and they were investigated by immunohistochemistry and liquid chromatography-tandem mass spectrometry. The interfaces were morphologically classified into three types: vertical, oblique, and convex. There were several cellular changes characteristic to the interface, including apoptosis and hyaline bodies, which were more emphasized in SCC/CIS sides. The results suggested that ND cells were winners of cell competition against SCC/CIS cells. Then, the interfaces were divided into four vertical segments, and each segment was separately laser-microdissected from tissue sections with immunostaining for K13 or K17; the four segments included SCC/CIS away from (#1) or adjacent to (#2) the interface, and ND adjacent to (#3) or away from (#4) the interface. Proteome analyses revealed approximately 4000 proteins from SCC/CIS sides [#1 and #2] and 2800 proteins from ND sides [#3 and #4]. We quantitatively selected the top 25 proteins including ladinin-1 or interleukin-1 receptor antagonist protein, which were most contrastively increased or decreased in SCC/CIS or ND sides, respectively, and their specific immunohistochemical expression modes were confirmed in tissue sections as well as in cultured SCC cells. These molecules should be involved in the cellular crosstalk toward cell competition at the lateral interface of oral SCC/CIS and would be new candidates for histopathological distinction of oral malignancies.

Simultaneous immunolocalization of desmoglein 3 and IgG4 in oral pemphigus vulgaris: IgG4 predominant autoantibodies in its pathogenesis.

BACKGROUND: Oral pemphigus vulgaris (PV), an autoimmune blistering disease, is mainly mediated by autoantibodies against desmoglein (Dsg) 3. However, no attention has been paid to IgG subclasses of the autoantibodies against Dsg3 in the diagnostic procedure for PV. Thus, our aim in this study was to investigate whether Dsg3 and any of IgG subclasses are immunohistochemically colocalized in tissue sections of PV oral mucosa. MATERIALS AND METHODS: Serial sections cut from formalin-fixed paraffin blocks of biopsy specimens of 9 PV cases and those of normal buccal mucosa surgically removed for fibro-epithelial polyps were comparatively examined for immunohistochemical localizations for Dsg3, IgG4, and IgG. RESULTS: Dsg3 was demonstrated in a dot-like pattern on the cell border and in the cytoplasm of the whole epithelial layer in both normal and PV specimens, while its staining was irregular among floating epithelial sheets of PV. IgG4 was also demonstrated in a punctuated fashion on the cell border among floating epithelial sheets, which was nearly identical to the immunohistochemical profile of Dsg3. In addition to being detected in the epithelial part, IgG4 signals were prominently localized in plasma cells scattered in the granulation tissue, where ratios of IgG4-positive (+) plasma cells to IgG+ cells were extraordinarily higher (mean 28%) than those in normal mucosa. DISCUSSION: These findings confirmed for the first time that autoantibodies against Dsg3 are mainly composed of IgG4 in oral PV and that the combined immunohistochemistry for Dsg3 and IgG4 can be a valuable aid in confirming a histopathological diagnosis of PV.

MFG-E8 expression for progression of oral squamous cell carcinoma and for self-clearance of apoptotic cells.

Milk fat globule--epidermal growth factor (EGF)--factor VIII (MFG-E8) is a secreted glycoprotein that promotes clearance of apoptotic cells by bridging phosphatidylserine on apoptotic cells and integrin αvß3/5 on phagocytes. High expression of MFG-E8 has been reported in various types of cancer in humans. Apoptotic figures are frequently found in the surgical samples of oral squamous cell carcinoma (SCC) and carcinoma in situ, and we have often observed apoptotic carcinoma cells engulfed by macrophages or even by neighboring carcinoma cells. Thus we hypothesized that MFG-E8 might promote engulfment of apoptotic carcinoma cells by living carcinoma cells and that MFG-E8 expressed by carcinoma cells could contribute to tumor progression. The aim of this study was to elucidate the biological role of MFG-E8 in oral SCC. Fifty-three surgical specimens of oral SCC were used for immunohistochemistry for MFG-E8, and the expression profiles were correlated with clinicopathological properties. Also, we examined the MFG-E8 expression patterns and functions using three human oral SCC cell lines. Most of the cases had MFG-E8-positive SCC cells, and the expression of MFG-E8 was correlated with such clinicopathological features as tumor size, pathological stage, locoregional recurrence, scattering invasion pattern, and SCC cell figures engulfing apoptotic SCC cells. The MFG-E8 staining was enhanced in apoptotic SCC cells, some of which were apparently engulfed by the neighboring SCC cells. ZK-1 cells showed high MFG-E8 expression, and its localization was found in the cytoplasm and the cell surface. Transient MFG-E8 knockdown by siRNA in ZK-1 decreased cell proliferation and invasiveness and increased cell death. Thus we have demonstrated that MFG-E8 promotes tumor progression in oral SCC and that it might be involved in the clearance of apoptotic SCC cells by living SCC cells.

Perlecan-enriched intercellular space of junctional epithelium provides primary infrastructure for leukocyte migration through squamous epithelial cells.

The aim of this study was to demonstrate the presence of intraepithelial stroma represented by extracellular matrix (ECM) deposits in the junctional epithelium to clarify its function as a scaffold for leukocyte migration through epithelial cells. Twenty-three biopsy specimens from the gingiva including the junctional epithelium were examined to determine comparative protein and gene level expression profiles for keratin and ECM molecules between the junctional epithelium and the gingival epithelium using immunohistochemistry and in situ hybridization. Intraepithelial leukocyte types and frequencies were also determined and compared between the junctional and gingival epithelia. In the junctional epithelium, which was positive for keratin 19, perlecan was strongly deposited in intercellular space of the whole epithelial layer, while it was faintly positive around the parabasal layer of the gingival epithelium. Perlecan mRNA signals were enhanced to a greater degree in both epithelial and inflammatory cells within the junctional epithelium. In the junctional epithelium, greater numbers of neutrophils and macrophages were found as compared with the gingival epithelium. Our results showed that perlecan is the primary ECM molecule comprising intraepithelial stroma of the junctional epithelium, in which leukocytes may migrate on ECM scaffolds in intercellular space toward the surface of the gingival sulci or pockets.

Keratin pearl degradation in oral squamous cell carcinoma: reciprocal roles of neutrophils and macrophages.

BACKGROUND: We have reported that neutrophilic infiltration was associated with round-shaped dyskeratosis foci, a kind of keratin pearl, of oral carcinoma in situ and that those inflammatory cells are recruited from intra-epithelially entrapped blood vessels. Based on these lines of evidence, we have formulated a hypothesis that keratin pearls are terminally degraded by neutrophils. To confirm this hypothesis, we investigated immunohistochemically stepwise degradation of keratin pearls in oral squamous cell carcinoma (SCC) to clarify any other type scavenger cells in addition to neutrophils are involved in this particular degradation process. METHODS: Neutrophils (neutrophil elastase) and macrophage subpopulations (CD68, CD163 and CD204) were immunohistochemically localized in 30 cases of oral SCC with typical round-shaped keratin pearls. SCC cells were revealed by immunohistochemistry for keratin (K) 17, and blood vessels were demonstrated by CD31. RESULTS: Keratin pearl degradation process was divided into four steps: (i) intact stage: no macrophage infiltration but minimal neutrophils were found in keratin pearls; (ii) neutrophil recruit stage: no macrophage infiltration but focal neutrophilic infiltration within the pearls; (iii) neutrophil predominant stage: dense neutrophil infiltration with minimal macrophages and segregated keratinized cancer cells strongly positive for K17; and (iv) macrophage predominant stage: dense infiltration of CD68-, CD163 (mononuclear)- and CD204 (multinucleated)-positive macrophages engulfing detached keratinized SCC cells. CONCLUSION: Keratin pearl degradation in oral SCC is strictly regulated by two types of scavenger cells: neutrophils, which perform initial tasks, and macrophages, which reciprocally take over from neutrophils the role to finalize the degradation processes.

Three-dimensional visualization of perlecan-rich neoplastic stroma induced concurrently with the invasion of oral squamous cell carcinoma.

BACKGROUND: We have demonstrated the induction of perlecan-rich stroma of oral squamous cell carcinoma (SCC) on and after its start of invasion. However, it remains unknown how such a neoplastic stroma is actually arranged in tumor tissues. METHODS: To this end, tissue microarray samples, in which keratin and perlecan were contrastively labeled by immunohistochemistry, were three-dimensionally analyzed using digital images and image analysis software to demonstrate the relationship between SCC foci and the perlecan-positive stromal space or that between carcinoma in situ (CIS) and invasive SCC foci. RESULTS: The three-dimensional (3D) reconstruction demonstrated three kinds of perlecan profiles for inside (I) and outside (O) areas of the carcinoma cell focus: mode 1, I(+)/O(-) ; mode 2, I(+)/O(+) ; and mode 3, I(-)/O(+). Mode 1 was seen in CIS as well as SCC tumor massifs in the surface part. Mode 2 was seen in small SCC foci, which seemed isolated in 2D sections but were mostly continuous with the tumor massif in 3D reconstructions. Mode 3 was limited to small SCC foci, which were truly segregated from the tumor massif. CONCLUSIONS: The results indicated that the 2D SCC focus isolation could not be regarded as invasion but that the SCC foci surrounded by perlecan-positive stroma (modes 2 and 3) could be regarded as a more objective measure for invasion of SCC. This is the first 3D tissue-level demonstration of the neoplastic stroma space induced with oral SCC invasion, the presence of which we have predicted based on our previous 2D and tissue culture evidence.

PAK4 inhibition augments anti-tumour effect by immunomodulation in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumours, warranting novel treatments. Here, we examined the therapeutic efficacy of inhibiting p21 activated kinase 4 (PAK4) in OSCC and determined its immunomodulatory effect by focusing on the enhancement of anti-tumour effects. We examined PAK4 expression in OSCC cells and human clinical samples and analysed the proliferation and apoptosis of OSCC cells following PAK4 inhibition in vitro. We also investigated the effects of in vivo administration of a PAK4 inhibitor on immune cell distribution and T-cell immune responses in OSCC tumour-bearing mice. PAK4 was detected in all OSCC cells and OSCC tissue samples. PAK4 inhibitor reduced the proliferation of OSCC cells and induced apoptosis. PAK4 inhibitor significantly attenuated tumour growth in mouse and was associated with increased proportions of IFN-γ-producing CD8+ T-cells. Furthermore, PAK4 inhibitor increased the number of dendritic cells (DCs) and up-regulated the surface expression of various lymphocyte co-stimulatory molecules, including MHC-class I molecules, CD80, CD83, CD86, and CD40. These DCs augmented CD8+ T-cell activation upon co-culture. Our results suggest that PAK4 inhibition in OSCC can have direct anti-tumour and immunomodulatory effects, which might benefit the treatment of this malignancy.

The effects of carbon-ion beam irradiation on three-dimensional in vitro models of normal oral mucosa and oral cancer: development of a novel tool to evaluate cancer therapy.

Given that the original tumor microenvironment of oral cancer cannot be reproduced, predicting the therapeutic effects of irradiation using monolayer cultures and animal models of ectopic tumors is challenging. Unique properties of carbon-ion irradiation (CIR) characterized by the Bragg peak exert therapeutic effects on tumors and prevent adverse events in surrounding normal tissues. However, the underlying mechanism remains unclear. The biological effects of CIR were evaluated on three-dimensional (3D) in vitro models of normal oral mucosa (NOMM) and oral cancer (OCM3 and OCM4) consisting of HSC-3 and HSC-4 cells. A single 10- or 20-Gy dose of CIR was delivered to NOMM, OCM3, and OCM4 models. Histopathological and histomorphometric analyses and labeling indices for Ki-67, γH2AX, and TUNEL were examined after CIR. The concentrations of high mobility group box 1 (HMGB1) were measured. NOMM exhibited epithelial thinning after CIR, which could be caused by the decreased presence of Ki-67-labeled basal cells. The relative proportion of the thickness of cancer cells to the underlying stroma in cancer models decreased after CIR. This finding appeared to be supported by changes in the three labeling indices, indicating CIR-induced cancer cell death, mostly via apoptosis. Furthermore, the three indices and the HMGB1 release levels significantly differed among the OCM4 that received different doses and with different incubation times after CIR while those of the OCM3 models did not, suggesting more radiosensitivity in the OCM4. The three 3D in vitro models can be a feasible and novel tool to elucidate radiation biology.

Rapamycin Induces Phenotypic Alterations in Oral Cancer Cells That May Facilitate Antitumor T Cell Responses.

OBJECTIVES: In this study, we investigated the antitumor immunomodulatory effects of rapamycin in oral cancer. STUDY DESIGN: We examined the proliferation, apoptosis, and migration of cancer cells and investigated the cell surface expression levels of immune accessory molecules and T cell immune responses in vitro. We investigated the effect of in vivo administration of rapamycin on immune cell distribution and T cell immune responses in oral tumor-bearing mice. RESULTS: Rapamycin treatment significantly inhibited OSCC cell proliferation and migration, increased apoptotic cell death, and upregulated cell surface expression of several immune accessory and adhesion molecules, including CD40, CD83, PD-L1, PD-L2, MHC class I, P-selectin, and VCAM-1. These cancer cells augmented T cell proliferation. In vivo rapamycin administration significantly attenuated mouse tumor growth with an increased proportion of immune cells, including CD4+ T cells, CD8+ T cells, and dendritic cells (DCs); decreased the proportion of immune suppressive cells, such as myeloid-derived suppressor cells and regulatory T cells; enhanced DC maturation and upregulated the surface expression of CD40, CD86, and ICAM-1. CONCLUSIONS: Our results suggest that the therapeutic effect of mTOR inhibition in oral cancer can cause direct antitumor and immunomodulatory effects.

Hemophagocytosis-mediated keratinization in oral carcinoma in situ and squamous cell carcinoma: a possible histopathogenesis of keratin pearls.

Although the histopathogenetic process of keratin pearls is still poorly understood, acceleration of keratinization in squamous cell carcinoma (SCC) cells may represent one possible therapeutic avenue. Based on our histopathological observations, we have hypothesized that SCC cells are keratinized by phagocytosis of extravasated erythrocytes. To confirm this hypothesis, we firstly examined immature keratin pearls in oral carcinoma in situ (CIS) and mature ones in SCC by immunohistochemistry. Concentric dyskeratotic cells in CIS keratin pearls became positive for keratin (K) 10, K17, heme oxygenase-1 (HO-1), or protease activated receptor-2 (PAR-2), a candidate regulator for hemophagocytosis. When ZK-1 cells, an SCC cell system, were incubated with human peripheral blood erythrocytes, or with crude and purified hemoglobins (Hbs), their erythro-hemophagocytotic activities were confirmed by immunofluorescence. Immunofluorescence signals for K10, K17, and HO-1 were enhanced due to hemophagocytosis in time-dependent manners. mRNA expression levels for the three molecules were most enhanced by purified Hb, followed by crude Hb and erythrocytes. K17/K10 mRNA expression levels were more elevated when PAR-2 was activated in ZK-1 cells. The results indicated that immature and mature keratin pearls in CIS and SCC were generated by oxidative stresses derived from erythro-hemophagocytosis, which might mediate HO-1 expression and be regulated by PAR-2. Thus, hemorrhage from the rupture of blood vessels can be one of the triggers for keratin pearl formation in oral CIS and SCC.

Podoplanin-mediated cell adhesion through extracellular matrix in oral squamous cell carcinoma.

Podoplanin (PDPN), one of the representative mucin-like type-I transmembrane glycoproteins specific to lymphatic endothelial cells, is expressed in various cancers including squamous cell carcinoma (SCC). On the basis of our previous studies, we have developed the hypothesis that PDPN functions in association with the extracellular matrix (ECM) from the cell surface side. The aim of this study was to elucidate the molecular role of PDPN in terms of cell adhesion, proliferation, and migration in oral SCC cells. Forty-four surgical specimens of oral SCC were used for immunohistochemistry for PDPN, and the expression profiles were correlated with their clinicopathological properties. Using ZK-1, a human oral SCC cell system, and five other cell systems, we examined PDPN expression levels by immunofluorescence, western blotting, and real-time PCR. The effects of transient PDPN knockdown by siRNA in ZK-1 were determined for cellular functions in terms of cell proliferation, adhesion, migration, and invasion in association with CD44 and hyaluronan. Cases without PDPN-positive cells were histopathologically classified as less-differentiated SCC, and SCC cells without PDPN more frequently invaded lymphatics. Adhesive properties of ZK-1 were significantly inhibited by siRNA, and PDPN was shown to collaborate with CD44 in cell adhesion to tether SCC cells with hyaluronan-rich ECM of the narrow intercellular space as well as with the stromal ECM. There was no siRNA effect in migration. We have demonstrated the primary function of PDPN in cell adhesion to ECM, which is to secondarily promote oral SCC cell proliferation.

Extracellular heat shock protein A9 is a novel interaction partner of podoplanin in oral squamous cell carcinoma cells.

In previous studies, we have shown several lines of evidence that podoplanin (PDPN) plays an important role in cell adhesion via its association with extracellular components in neoplastic conditions, though there has been no trial to search for PDPN-interaction molecules in the extracellular milieu. To screen for those molecules, we performed proteomics-based analysis using liquid chromatography-tandem mass spectrometry followed by co-immunoprecipitation for PDPN in ZK-1, an oral squamous cell carcinoma (SCC) cell system whose cell membrane molecules were cross-linked with each other in their extracellular compartments, and we identified heat shock protein (HSP) A9 as one of the extracellular PDPN bound molecules. Effects of transient PDPN knockdown by siRNA in ZK-1 were also comparatively examined for cellular behaviors in terms of HSPA9 expression and secretion. Finally, HSPA9 expression modes were immunohistochemically visualized in oral SCC tissue specimens. HSPA9 was secreted from ZK-1 cells, and the expression and secretion levels of HSPA9 gene and protein were well coordinated with those of PDPN. Immunohistochemically, HSPA9 and PDPN were co-localized in ZK-1 cells and oral SCC foci, especially in the peripheral zone. In conclusion, the results indicate that HSPA9 secreted by oral SCC cells interacts with PDPN on their cell surface in an autocrine manner and regulates their growth and invasiveness.

Identification of a soluble isoform of human IL-17RA generated by alternative splicing.

IL-17RA, a member of the interleukin (IL)-17 receptor family, is a single membrane-spanning protein that ubiquitously expressed on the cell surface. IL-17RA transduces IL-17A, IL-17F, and IL-17A/F heterodimer-mediated signals by forming a complex with IL-17RC, and also signals the IL-17E (also known as IL-25) response in combination with IL-17RB (also known as IL-25R). Previously, soluble isoforms of human IL-17RC and IL-17RB have been reported, but the existence of a soluble isoform of human IL-17RA has remained unclear. Here, we report the identification of a soluble isoform of human IL-17RA at the mRNA and protein levels. Reverse transcribed PCR experiments showed that the IL-17RA variant is generated by spliced out of exon 11 encoding the transmembrane region in a variety of human tissues. The soluble IL-17RA isoform was detected in the culture media of human cell lines by Western blotting. The existence of the soluble IL-17RA isoform sheds new light on the regulation of IL-17RA mediated responses.