Molecular biological role of epithelial splicing regulatory protein 1 in intrahepatic cholangiocarcinoma.

AIM: Epithelial splicing regulatory protein 1 (ESRP1) regulates tumor progression and metastasis through the epithelial‒mesenchymal transition by interacting with zinc finger E-box binding 1 (ZEB1) and CD44 in cancers. However, the role of ESRP1 in intrahepatic cholangiocarcinoma (iCCA) remains unclear. METHODS: Three iCCA cell lines (HuCCT-1, SSP-25, and KKU-100) were analyzed using small interfering RNA to investigate the molecular biological functions of ESRP1 and ZEB1. The association between clinicopathological features and the expression of ESRP1 and ZEB1 in iCCA tissues was analyzed immunohistochemically. Proteomic analysis was performed to identify molecules related to ESRP1 expression. RESULTS: ESRP1 expression was upregulated in HuCCT-1 and SSP-25 cells. Cell migration and invasion were enhanced, and the expression of ZEB1 and CD44s (CD44 standard) isoforms were upregulated in the ESRP1 silencing cells. Moreover, ESRP1 silencing increased the expression of N-cadherin and vimentin, indicating the presence of mesenchymal properties. Conversely, ZEB1 silencing increased the expression of ESRP1 and CD44v (CD44 variant) isoforms. Immunohistochemical analysis revealed that a lower ESRP1-to-ZEB1 expression ratio was associated with poor recurrence-free survival in patients with iCCA. Flotillin 2, a lipid raft marker related to epithelial‒mesenchymal transition, was identified as a protein related to the interactive feedback loop in proteomic analysis. CONCLUSIONS: ESRP1 suppresses tumor progression in iCCA by interacting with ZEB1 and CD44 to regulate epithelial‒mesenchymal transition.

2-Deoxy-d-glucose increases GFAT1 phosphorylation resulting in endoplasmic reticulum-related apoptosis via disruption of protein N-glycosylation in pancreatic cancer cells.

The glycolytic inhibitor 2-deoxy-d-glucose (2DG) causes energy starvation, affecting cell viability in a wide range of cancer cell lines. To determine the action of 2DG in pancreatic cancer, we performed proteomic analysis of pancreatic cancer cell line after 2DG treatment. Eighty proteins showed differential expression and among these, proteins involved in phosphohexose metabolism were upregulated. Up-regulation of glutamine: fructose 6-phosphate aminotransferase 1 (GFAT1), which belongs to the hexosamine biosynthesis pathway (HBP) that produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to maintain glycoprotein, was validated by evaluation of mRNA and protein levels. Therefore, we assessed the amounts of total N-glycoproteins. Unexpectedly, we found a reduction of total N-glycoproteins and phosphorylation of GFAT1 by AMP-activated protein kinase (AMPK). These data may shed light on HBP dysfunction. Furthermore, we found endoplasmic reticulum (ER) stress accompanied by increased expression of ER stress markers, such as glucose response protein 78 (GRP78) and C/EBP-homologous protein (CHOP), in 2DG-treated cells. Moreover, the additive activation of AMPK by metformin (Met) synergistically enhanced the reduction of protein N-glycosylation and cell growth inhibition in the presence of 2DG. These results suggest that 2DG reduces N-glycosylation of proteins following the increase of phosphorylation of GFAT1 and results in the inhibition of cell growth mediated by ER stress in pancreatic cancer cells.

Identification of aldolase A as a potential diagnostic biomarker for colorectal cancer based on proteomic analysis using formalin-fixed paraffin-embedded tissue.

Colorectal cancer (CRC) is one of the most common cancers worldwide, and many patients are already at an advanced stage when they are diagnosed. Therefore, novel biomarkers for early detection of colorectal cancer are required. In this study, we performed a global shotgun proteomic analysis using formalin-fixed and paraffin-embedded (FFPE) CRC tissue. We identified 84 candidate proteins whose expression levels were differentially expressed in cancer and non-cancer regions. A label-free semiquantitative method based on spectral counting and gene ontology (GO) analysis led to a total of 21 candidate proteins that could potentially be detected in blood. Validation studies revealed cyclophilin A, annexin A2, and aldolase A mRNA and protein expression levels were significantly higher in cancer regions than in non-cancer regions. Moreover, an in vitro study showed that secretion of aldolase A into the culture medium was clearly suppressed in CRC cells compared to normal colon epithelium. These findings suggest that decreased aldolase A in blood may be a novel biomarker for the early detection of CRC.

Immunohistochemical Diagnosis of Amyloid Typing: Utility and Limitations as Determined by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Although immunohistochemical techniques and proteomic analysis are widely used for typing diagnosis of amyloidosis, the diagnostic utility of immunohistochemical evaluation is not well understood. METHODS: We used immunohistochemical techniques to characterize staining patterns of in-house rabbit polyclonal anti-κ, anti-λ, anti-transthyretin antibodies, and commercial anti-amyloid A and anti-ß2-microglobulin antibodies in 40 autopsy cases. RESULTS: In thirty cases (75%), the subtype was determined by using the criterion that amyloid is strongly and diffusely positive for one antibody while negative for other antibodies. We then performed proteomic analysis of all 40 cases. In 39 cases, we identified only one amyloid protein and confirmed the immunohistochemically determined subtypes of the abovementioned 30 cases. In seven other cases, we could retrospectively determine subtypes with immunohistochemistry by using information from proteomic analysis, which increased the immunohistochemistry diagnosis rate to 92.5% (37/40). In one case, we identified double subtypes, both immunohistochemically and with proteomic analysis. In the remaining three cases, proteomic analysis was essential for typing diagnosis. CONCLUSIONS: The present findings suggest that combined immunohistochemistry and proteomic analysis is more useful than immunohistochemistry alone. Our findings highlight the importance of carefully interpreting immunohistochemistry for anti-TTR and light chain and offer insights that can guide amyloid typing through immunohistochemistry.

Cytological Assessment of Desmoplastic Malignant Pleural Mesothelioma in an Autopsy Case.

INTRODUCTION: Desmoplastic malignant pleural mesothelioma (DMPM) is a sarcoma-type mesothelioma, comprising approximately 5% of malignant pleural mesotheliomas. Although effusion cytology is commonly used as the primary diagnostic approach for mesothelioma, it may not be useful for DMPM because of the presence of desmoplasia and bland cellular atypia. We report a case, and previously undescribed cytological features, of DMPM that was diagnosed during autopsy. CASE PRESENTATION: A man in his 60s with a history of occupational asbestos exposure was referred to our hospital with right chest pain. A chest CT scan showed right pleural effusion. Thirteen months later, the patient died of respiratory failure. During autopsy, scrape-imprint smears were prepared and cytology of pleural effusions was performed. The scrape-imprint smear samples showed spindle cells with mild nuclear atypia and grooves with fibrous stroma. Pleural effusion cytology revealed spindle cells with mild nuclear atypia, as well as grooves with loose epithelial connections. Histological examination of the right pleura showed spindle cells proliferating with dense collagen fibers, as seen in the cytological samples, thus indicating a diagnosis of DMPM, which was confirmed by fluorescence in situ hybridization. CONCLUSION: Cytological procedures such as pleural effusion cytology and scrape-imprinting cytology may help in diagnosing rare tumors such as DMPM.

Toll­like receptor 4 plays a tumor­suppressive role in cutaneous squamous cell carcinoma.

Toll­like receptor 4 (TLR4), a key regulator of the innate immune system, is expressed not only in immune cells, but also in a number of cancer cells. A biological role for TLR4 in cutaneous squamous cell carcinoma (SCC), however, is unclear. In this study, we first examined TLR4 expression and localization in cases of SCC, actinic keratosis (AK) and Bowen's disease (BD) by immunohistochemistry. TLR4 expression was significantly higher in the SCC than in the AK or BD tissues. We then determined the TLR4 expression level in vivo, in 3 histological subtypes of SCC. TLR4 expression in poorly differentiated SCC was significantly lower compared with that of the moderately and well­differentiated type. In addition, the CD44 immunoreactivity tended to be high in the cell membrane of poorly differentiated SCC. Of note, poorly differentiated SCC is a risk factor of unfavorable outcomes in affected patients. We then assessed the biological role of TLR4 in HSC­1 and HSC­5 SCC cells and HaCaT human keratinocytes. TLR4 knockdown by transfection with siRNA accelerated HSC­1 and HaCaT cell migration and invasion compared to the control siRNA­transfected cells. TLR4 knockdown resulted in an increased CD44 expression and in an enhanced filopodia protrusion formation, particularly in HSC­1. On the whole, these results suggest that a reduced TLR4 expression enhances the malignant features in SCC cases and cultured SCC cell lines. TLR4 may thus play an anti­tumor role in cutaneous SCC.

Insulin-like growth factor 2 mRNA-binding protein-3 as a marker for distinguishing between cutaneous squamous cell carcinoma and keratoacanthoma.

In the histopathological diagnosis of cutaneous tumors, the differential diagnosis of squamous cell carcinoma (SCC) with crateriform architecture and keratoacanthoma (KA) is often difficult so an accurate understanding of the biological features and the identification of reliable markers of SCC and KA are crucial issues. Insulin-like growth factor 2 mRNA-binding protein-3 (IGF2BP3, also known as IMP3) is thought of as a bona fide oncofetal protein, which is overexpressed and is involved in cell proliferation, migration, and invasion in several kinds of tumors. However, the role of IMP3 in cutaneous SCC and KA has not been well studied. Therefore, we focused on studying the biological functions of IMP3 in SCC and KA. In human skin SCC cell lines, HSC-1 and HSC-5, and the human keratinocyte cell line, HaCaT, IMP3 mRNA levels were significantly higher than that of normal human skin. The knockdown of IMP3 expression reduced the proliferation of HSC-1, and significantly reduced invasion by HSC-1 and HSC-5. In contrast, the knockdown of IMP3 did not significantly affect invasion by HaCaT cells. In immunohistochemical studies of SCC and KA tissues, the Ki-67 labeling index (LI) of the suprabasal cell layer was significantly higher in SCC, compared with KA tissues and the tumor-free margin (TFM) adjacent to SCC and KA. Most SCC tissues stained strongly positive for IMP3, but KA tissues and TFM were mostly negative for IMP3. The Ki-67 LI of the IMP3-positive group was significantly higher than that of the IMP3-negative group in the suprabasal cell layer of SCC. These results suggest that IMP3 plays an important role in proliferation and, more significantly, in the invasion of SCC, and may be a suitable marker for the histopathological diagnosis of SCC with a crateriform architecture and KA. Furthermore, IMP3 may potentially be a new therapeutic target for SCC.

Expression and accumulation of lumican protein in uterine cervical cancer cells at the periphery of cancer nests.

Lumican is a member of a small leucine-rich proteoglycan (SLRP) family and is reported to be overexpressed during the wound healing process of the cornea, and ischemic and reperfused heart. In the carcinomatous tissues, lumican is overexpressed in human breast and pancreatic cancer tissues. In the present study, we aimed to clarify the expression of lumican mRNA and its protein in human cervical cancer cell lines (CaSki, ME-180 and HeLa cells) and their localization in normal and cancerous human cervical tissues. Reverse transcription-polymerase chain reaction and Western blot analysis revealed the expression of lumican mRNA and its protein in CaSki, ME-180 and HeLa cells. No or weak immunoreactivity of the lumican protein was observed in stroma but not in squamous and ductal cells of non-cancerous uterine cervical tissues. In 21 of 28 (75%) cervical cancer cases, the lumican protein was strongly expressed in cancer cells, and accumulated particularly in cancer cells at the periphery of the cancer nests. It was also expressed in the fibroblasts adjacent to the cancer cells. In situ hybridization analysis revealed that lumican mRNA was not expressed in squamous or ductal epithelial cells in non-cancerous tissues, but was expressed in most cancer cells and stromal fibroblasts in uterine cervical cancer tissues. The lumican protein was not localized in normal squamous or ductal cells close to cancer cells, but its mRNA was strongly expressed in the same cells. To our knowledge, this is the first report on lumican synthesized by squamous cell carcinomas. These findings may indicate that the accumulated lumican protein in cancer cells at the periphery of cancer nests may play roles in the growth or invasion of human cervical cancer cells.

Altered expression of fibroblast growth factor receptor 2 isoform IIIc: relevance to endometrioid adenocarcinoma carcinogenesis and histological differentiation.

Fibroblast growth factor receptor 2 (FGFR2) is activated in many cancers and considered as a potential therapeutic molecular target including for endometrial endometrioid carcinoma (EEC). Overexpression of FGFR2 isoform IIIc (FGFR2IIIc) has been shown to be associated with carcinogenesis in various cancers, but its expression in EEC has not been reported yet to the best of our knowledge. In this study, we identified roles for FGFR2IIIc in EEC carcinogenesis and demonstrated its diagnostic and prognostic values in EEC. FGFR2IIIc expression was compared between 10 normal endometrium, 10 atypical endometrial hyperplasias, and 47 EEC specimens using immunohistochemistry and quantitative real-time PCR. Atypical hyperplasia, Grade 1 (G1), and Grade 2 (G2) differentiated EEC tissues showed significantly higher FGFR2IIIc expression than normal endometrium tissue. However, as compared to G1 and G2 EECs, Grade 3 (G3) differentiated EEC tissue showed lower FGFR2IIIc expression (P<0.05). There was no significant association between FGFR2IIIc expression and patient age, lymph node metastasis, and EEC stage. These results suggest that altered FGFR2IIIc expression plays an important role in EEC carcinogenesis and may occur in precancerous tissues. However, FGFR2IIIc appears to be not related to EEC progression. Some G3 EECs may develop through different carcinogenic processes than G1 and G2 EECs.

Nodular fasciitis in the parotid gland: a case report and review of the literature.

Nodular fasciitis (NF) is a benign, reactive lesion with a self-limiting process. Because NF is rare in the parotid gland and has many cytological similarities to other benign or malignant tumors, cytological misinterpretation is common. The patient, a 30-year-old woman, had a painless mass in her right parotid gland. Fine needle aspiration cytology (FNAC) was performed. Spindle cells with basophilic and well-demarcated cytoplasm were observed in a mucoid-like background. The mucoid-like substance was metachromatic and appeared to be the matrix of PA. Histopathologically, spindle-shaped cells with intervening birefringent mature collagen were arranged in short irregular bundles. Prominent mucoid-like matrixes as well as few infiltrating neutrophils and lymphocytes were found in the background. Lesional cells were positive for CD10 and ß-catenin in the cytoplasm, but negative for cytokeratin, the S-100 protein, CD34, and neurofilament. Ultimately, this patient was diagnosed with NF. In FNAC of the parotid gland region, distinguishing NF from other real tumors is important for deciding treatment strategies.

Neuroepithelial stem cell marker nestin regulates the migration, invasion and growth of human gliomas.

Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell marker during central nervous system development. Nestin is expressed in gliomas, and its expression levels are higher in gliomas with high WHO histopathological classification grades than in those with low grades. In the present study, we examined whether nestin regulates the biological activities of human glioma cells. Immunohistochemically, the nestin expression patterns in 10 human glioblastoma patients were examined. The expression levels of nestin in A172, a human high-grade glioma cell line, and KG-1-C, a human low-grade glioma cell line, were examined using real-time PCR, Western blot and immunofluorescence analyses. An expression vector carrying a short hairpin RNA targeting nestin was stably transfected into A172 (Sh) cells. The effects of decreased expression levels of nestin in Sh cells on cell growth, migration, invasion, adhesion to extracellular matrices and fibrillar actin expression on three-dimensional culture plates were examined. The nestin expression vector was transiently transfected into KG-1-C (Nes) cells, and the effects of the nestin overexpression on cell growth and migration were examined. Nestin was expressed in the cytoplasm of the glioblastoma cells in all cases examined. Sh cells showed marked decreases in the expression levels of nestin mRNA and protein, and the growth rate of Sh cells was lower than that of sham (Sc) cells. In contrast, the adhesion activity of Sh cells to types I and IV collagens, fibronectin and laminin was higher than that of Sc cells. Fibrillar actin was clearly detected at the periphery of colonies of Sh cells at the attachment sites on three-dimensional culture plates. The migration and invasion of Sh cells were markedly inhibited compared with those of Sc cells. In contrast, the levels of nestin expression markedly increased in the Nes cells, which were transiently transfected with the nestin expression vector. The growth rate and motility of Nes cells were higher than those of the mock cells. In conclusion, nestin plays important roles in cell growth, migration, invasion and adhesion to extra-cellular matrices in glioma cells. Nestin may serve as a novel candidate for molecular-targeted therapy for gliomas, including glioblastomas.

Morphological and cytoskeletal alterations of nervous system tumor cells with different culturing methods.

Cell culture is one of the most important methods of research in molecular and cellular biology, and various culture systems have been developed, including two-dimensional (2D), three-dimensional (3D) and floating culture systems. In the present study, we examined morphological changes and different expression patterns of cytoskeletal proteins in three different types of nervous system tumor cells grown in 2D, 3D and floating cell cultures. A172, KG-1-C and IMR-32 cells showed marked morphological changes, depending on the cell culture methods. F-actin expression was clearly observed at the level of the cells nearest the plate surface in 2D and 3D cultures. On the other hand, expression of F-actin was weak in the floating culture system. α-tubulin was detected in the cytoplasm of cells in 2D culture, but in floating and 3D cultures, α-tubulin was expressed in the peripheral regions of spheres and spheroids. In conclusion, this study demonstrated that nervous system tumor cells showed different alterations in morphology, and different cytoskeletal protein expression patterns, depending on the culture methods.

Comparison of fixation methods for preservation of morphology, RNAs, and proteins from paraffin-embedded human cancer cell-implanted mouse models.

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.

Keratinocyte growth factor-transfection-stimulated adhesion of colorectal cancer cells to extracellular matrices.

The keratinocyte growth factor (KGF) regulates cell growth and behavior in an autocrine or paracrine manner. In colorectal cancer tissues, KGF is expressed in tumor cells and adjacent stromal fibroblasts. We have constructed a KGF-gene-transfected cell line (HCT15-KGF) from a colorectal cancer cell line, HCT-15, that expresses the KGF receptor, and studied the effects of KGF on cell behavior, particularly growth and adhesion to extracellular matrices (ECMs). The amount of KGF secreted from HCT15-KGF was significantly higher than that from a mock-transfected cell line (HCT15-MOCK). The modes of growth of these cell lines were similar. The degree of adhesion of HCT15-KGF to ECMs, including type-IV collagen and fibronectin was higher than that of HCT15-MOCK. The expressions of integrins in both cell lines were not significantly different. However, extracellular-regulated kinase-1 and -2 (ERK1/2) phosphorylation and focal adhesion kinase (FAK) expression that regulate the adhesive functions of integrin families were enhanced in HCT15-KGF. U0126, an inhibitor of the ERK upstream regulator MEK, attenuated the adhesion and spreading of HCT15-KGF cells to type-IV collagen. These results indicate that KGF enhances the adhesion of colorectal cancer cells to type-IV collagen through ERK and FAK signaling pathways.

Defined localization of nestin-expressing cells in L-arginine-induced acute pancreatitis.

OBJECTIVE: Nestin is a stem cell marker originally described as an intermediate filament protein expressed in neuroepithelial stem cells. In the pancreas, a small number of nestin-expressing cells, which are believed to represent either stem cells or progenitor cells, are known to be present in islets, as well as in some stellate cells, pericytes, and endothelial cells. We monitored pancreatic nestin expression to delineate the location of stem cells/progenitor cells in the pancreas after L-arginine-induced pancreatitis. METHODS: Male Wistar rats received 2 intraperitoneal injections of L-arginine, each consisting of 250 mg/100 g of body weight, and were killed 3, 6, and 12 hours and 1, 4, 7, and 14 days later. RESULTS: Serum amylase and lipase levels increased after L-arginine injection, maximal levels occurring at 3 and 12 hours postinjection, respectively. Six hours after L-arginine injection, interstitial edema was observed in the pancreas, whereas on day 4 postinjection, there was severe pancreatic necrosis. Neovascularization and ductal-ductular proliferation were also present in the pancreas. Immunohistochemical analysis revealed increased Ki-67 labeling in acinar cells and capillary endothelial cells. Immunoblotting using antinestin antibody revealed increased nestin expression after L-arginine injection. In the control rat pancreas, nestin immunoreactivity was detected in a few capillary endothelial cells in some islets. After L-arginine injection, nestin was expressed in proliferating capillary endothelial cells, in stellate cells surrounding ductular structures and in submesothelial cells. CONCLUSIONS: Transient nestin expression occurs in specific cell types during the proliferative stage after recovery from L-arginine-induced pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the regenerative capacity of the pancreas.

Differential distribution of fibroblast growth factor (FGF)-7 and FGF-10 in L-arginine-induced acute pancreatitis.

The regenerative process of the pancreas after acute pancreatitis (AP) is characterized by acinar and ductal cell proliferation with synthesis and transient deposition of extracellular matrices. Various growth factors were reported to be highly expressed in AP, but their regulation has not yet been clarified. Fibroblast growth factor (FGF)-7, also known as keratinocyte growth factor (KGF), and FGF-10 are members of the FGF family and show high structural homology and similar biological characteristics. Both are mainly synthesized by mesenchymal cells and stimulate epithelial cells via KGF receptor (KGFR) which is a splice variant of FGFR-2. In the present study, we attempted to immunohistochemically determine the localization of FGF-7 and FGF-10 in pancreatic tissues of an L-arginine-induced rat pancreatitis model. Furthermore, highly specific KGFR antibodies were prepared and used for Western blot analysis and immunohistochemistry. In the normal pancreas, FGF-7 was localized in alpha cells of islets, but FGF-10 was not detected. KGFR was also localized in islet cells, ductal cells, and centroacinar cells in the normal pancreas. In the pancreatic tissues of rats with L-arginine-induced pancreatitis, FGF-7 was localized in alpha cells, whereas FGF-10 was expressed in vascular smooth muscle cells (VSMCs). KGFR was not expressed in centroacinar cells and its level decreased after L-arginine treatment. However, KGFR was detected instead in some acinar cells and VSMCs in addition to islet cells. These findings suggest that FGF-7 and FGF-10 contribute to the regeneration and differentiation of acinar cells and angiogenesis in AP through KGFR.

Transient and ectopic expression of lumican by acinar cells in L-arginine-induced acute pancreatitis.

Lumican is a member of a small leucine-rich proteoglycan family. We previously found that lumican mRNA and its protein were ectopically and highly expressed in acinar cells in chronic pancreatitis (CP)-like lesions close to pancreatic cancer cells. CP-like lesions are characterized by acinar and ductal-ductular cell proliferation with expanding fibrosis. This finding suggests that lumican is ectopically synthesized by acinar cells under chronic inflammatory conditions and plays a role in fibrosis of the pancreas. However, the expression and role of lumican in acute inflammatory changes of the pancreas are not completely elucidated. In the present study, we aim to clarify whether lumican mRNA and its protein are expressed in exocrine or endocrine components in acute pancreatitis (AP). For experimental AP, Wistar rats received an intraperitoneal injection of L-arginine. Western blot analysis showed an intense 50-kDa band corresponding to the lumican protein in normal and L-arginine-treated rat pancreas. After L-arginine injection, three intense bands at 42, 57, and 92 kDa were detected on day 1. Immunohistochemically, the lumican protein was localized in ductal and a few centroacinar cells in the normal pancreas. After L-arginine injection, an immature fibrosis with fragmented and loose collagen fibers was observed in AP on day 4 and lumican immunoreactivity was detected in the collagen fibers. Lumican mRNA was faintly detected in islet cells in the normal pancreas, but it was strongly expressed in acinar and islet cells on day 1. Furthermore, lumican mRNA was expressed in many proliferating fibroblasts on day 4 by in situ hybridization. These findings indicate that lumican is transiently synthesized by acinar cells and fibroblasts in AP. Lumican proteins synthesized by acinar cells, islet cells, and fibroblasts may contribute to immature and transient fibrosis of AP.

Different influences of hyperglycemic duration on phosphorylated extracellular signal-regulated kinase 1/2 in rat heart.

Extracellular signal-regulated kinase (ERK) 1/2 is an important intracellular proteinase associated with myocardial protection against heart injury. Hyperglycemia was also reported to be highly involved in heart injury by the formation of advanced glycation end products (AGEs) in myocardial protein, resulting in its altered structure and function. However, the effect of this glycation on mitogen-activated protein kinases, particularly ERK1/2, in the myocardium is largely unclarified. In this study, we investigated whether the glycation of an intracellular protein, ERK1/2, would result in ERK1/2-AGEs formation that adversely affects ERK1/2 activation in the rat heart under hyperglycemia. Hyperglycemia was induced by injection of streptozotocin (STZ) and hearts were examined 4 and 20 weeks after STZ treatment. By immunohistochemical staining and Western blotting, it was determined that the level of phosphorylated ERK1/2 in the rat heart under hyperglycemia 20 weeks after STZ treatment decreased markedly by about 50% of that of the time-matched control group, whereas in the case of 4 weeks after STZ treatment, it increased by about 2.7-fold that of the time-matched group. The level of deposition of AGEs in proteins of the myocardium increased significantly depending on the duration of hyperglycemia. Twenty weeks after STZ treatment, two clear bands corresponding to 44- and 42-kDa AGEs were detected by Western blotting: these corresponded to protein sizes of ERK1/2. The immunoprecipitation method further confirmed the formation and the increased intensity of ERK1/2-AGEs in the rat heart under hyperglycemia for 20 weeks. These results demonstrate that long-term hyperglycemia may inhibit ERK1/2 phosphorylation in the myocardium, whereas a short-term (4 weeks) hyperglycemia enhances its phosphorylation. The ERK1/2 phosphorylation under long-term hyperglycemia is very different from that under short-term hyperglycemia. In addition, this inhibition of ERK1/2 activation appears to be dependent on the formation of ERK1/2-AGEs under long-term hyperglycemia, which may be related in part to the etiology of diabetic cardiomyopathy. It also suggests that the formation of AGEs in intracellular enzymes and proteins under hyperglycemia could play important roles in the development of diabetes complications.