Acid-sensing receptor GPR4 plays a crucial role in lymphatic cancer metastasis.

Cancer tissues exhibit an acidic microenvironment owing to the accumulation of protons and lactic acid produced by cancer and inflammatory cells. To examine the role of an acidic microenvironment in lymphatic cancer metastasis, gene expression profiling was conducted using human dermal lymphatic endothelial cells (HDLECs) treated with a low pH medium. Microarray and gene set enrichment analysis revealed that acid treatment induced the expression of inflammation-related genes in HDLECs, including genes encoding chemokines and adhesion molecules. Acid treatment-induced chemokines C-X3-C motif chemokine ligand 1 (CX3CL1) and C-X-C motif chemokine ligand 6 (CXCL6) autocrinally promoted the growth and tube formation of HDLECs. The expression of vascular cell adhesion molecule 1 (VCAM-1) increased in HDLECs after acid treatment in a time-dependent manner, which, in turn, enhanced their adhesion to melanoma cells. Among various acid-sensing receptors, HDLECs basally expressed G protein-coupled receptor 4 (GPR4), which was augmented under the acidic microenvironment. The induction of chemokines or VCAM-1 under acidic conditions was attenuated by GPR4 knockdown in HDLECs. In addition, lymph node metastases in a mouse melanoma model were suppressed by administering an anti-VCAM-1 antibody or a GPR4 antagonist. These results suggest that an acidic microenvironment modifies the function of lymphatic endothelial cells via GPR4, thereby promoting lymphatic cancer metastasis. Acid-sensing receptors and their downstream molecules might serve as preventive or therapeutic targets in cancer.

The Gain-of-Function Mutation p53R248W Suppresses Cell Proliferation and Invasion of Oral Squamous Cell Carcinoma through the Down-Regulation of Keratin 17.

Keratin 17 (KRT17) expression promotes the proliferation and invasion of oral squamous cell carcinoma (OSCC), and mutations in TP53 have been reported in 65% to 85% of OSCC cases. We studied the correlation between KRT17 expression and TP53 mutants. Ca9-22 cells, which exhibit low KRT17 expression, carried mutant p53 (p53R248W) and p53R248W knockdown promoted KRT17 expression. p53R248W knockdown in Ca9-22 cells promoted migration and invasion activity. In contrast, in HSC3 cells, which have p53 nonsense mutations and exhibit high KRT17 expression, the overexpression of p53R248W decreased KRT17 expression, cell size, proliferation, and migration and invasion activities. In addition, p53R248W significantly suppressed MMP2 mRNA expression and enzyme activity. Moreover, s.c. and orthotopic xenografts were generated from p53R248W- or p53R248Q-expressing HSC3 cells. Tumors formed from p53R248W-expressing HSC3 cells grew more slowly and had a lower Ki-67 index than those derived from the control or p53R248Q-expressing HSC3 cells. Finally, the survival rate of the mice inoculated with p53R248W-expressing HSC3 cells was significantly higher than that of the control mice. These results indicate that the p53R248W mutant suppresses proliferation and invasion activity through the suppression of KRT17 expression. We propose that OSCC with p53R248W-expressing cells may be classified as a new OSCC type that has a good prognosis.

Smad3 Suppresses Epithelial Cell Migration and Proliferation via the Clock Gene Dec1, Which Negatively Regulates the Expression of Clock Genes Dec2 and Per1.

Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression.

Dec1 Deficiency Suppresses Cardiac Perivascular Fibrosis Induced by Transverse Aortic Constriction.

Cardiac fibrosis is a major cause of cardiac dysfunction in hypertrophic hearts. Differentiated embryonic chondrocyte gene 1 (Dec1), a basic helix-loop-helix transcription factor, has circadian expression in the heart; however, its role in cardiac diseases remains unknown. Therefore, using Dec1 knock-out (Dec1KO) and wild-type (WT) mice, we evaluated cardiac function and morphology at one and four weeks after transverse aortic constriction (TAC) or sham surgery. We found that Dec1KO mice retained cardiac function until four weeks after TAC. Dec1KO mice also revealed more severely hypertrophic hearts than WT mice at four weeks after TAC, whereas no significant change was observed at one week. An increase in Dec1 expression was found in myocardial and stromal cells of TAC-treated WT mice. In addition, Dec1 circadian expression was disrupted in the heart of TAC-treated WT mice. Cardiac perivascular fibrosis was suppressed in TAC-treated Dec1KO mice, with positive immunostaining of S100 calcium binding protein A4 (S100A4), alpha smooth muscle actin (αSMA), transforming growth factor beta 1 (TGFß1), phosphorylation of Smad family member 3 (pSmad3), tumor necrosis factor alpha (TNFα), and cyclin-interacting protein 1 (p21). Furthermore, Dec1 expression was increased in myocardial hypertrophy and myocardial infarction of autopsy cases. Taken together, our results indicate that Dec1 deficiency suppresses cardiac fibrosis, preserving cardiac function in hypertrophic hearts. We suggest that Dec1 could be a new therapeutic target in cardiac fibrosis.

Potential Roles of Dec and Bmal1 Genes in Interconnecting Circadian Clock and Energy Metabolism.

The daily rhythm of mammalian energy metabolism is subject to the circadian clock system, which is made up of the molecular clock machinery residing in nearly all cells throughout the body. The clock genes have been revealed not only to form the molecular clock but also to function as a mediator that regulates both circadian and metabolic functions. While the circadian signals generated by clock genes produce metabolic rhythms, clock gene function is tightly coupled to fundamental metabolic processes such as glucose and lipid metabolism. Therefore, defects in the clock genes not only result in the dysregulation of physiological rhythms but also induce metabolic disorders including diabetes and obesity. Among the clock genes, Dec1 (Bhlhe40/Stra13/Sharp2), Dec2 (Bhlhe41/Sharp1), and Bmal1 (Mop3/Arntl) have been shown to be particularly relevant to the regulation of energy metabolism at the cellular, tissue, and organismal levels. This paper reviews our current knowledge of the roles of Dec1, Dec2, and Bmal1 in coordinating the circadian and metabolic pathways.

PRG4 expression in myxoid liposarcoma maintains tumor cell growth through suppression of an antitumor cytokine IL-24.

PRG4 is one of the downstream molecules of the myxoid liposarcoma (MLS)-specific fusion oncoproteins TLS-CHOP and EWS-CHOP. Exogenous PRG4 expression increases the tumorigenicity of cells injected in nude mice. The molecular functions of PRG4 in tumorigenesis and/or tumor progression of MLS cells, however, still remain unclear. In this report, we demonstrated that siRNA-mediated knockdown of PRG4 suppressed the growth of the MLS-derived cell lines 1955/91 and 2645/94. In addition, PRG4 knockdown promoted adipocytic differentiation in 1955/91 cells. Thus, PRG4 may play essential roles in MLS cell growth and have potential as a therapeutic target. On the other hand, our previous study has revealed that TLS-CHOP suppresses expression of an anti-tumor cytokine IL-24, contributing to tumor cell survival. In this study, we found that double knockdown of PRG4 and IL-24 did not inhibit MLS cell growth, and single knockdown of PRG4 remarkably increased IL-24 expression. These results suggest that the growth inhibitory effect of PRG4 knockdown is caused by induction of IL-24 expression, and PRG4 may contribute to maintain MLS cell growth through repression of IL-24 expression.

Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts via TNF-α.

Bmal1, a clock gene, is associated with depression, hypertrophy, metabolic syndrome and diabetes. Smad3, which is involved in the TGF-ß signaling pathway, plays an important role in the regulation of tumor progression, fibrosis, obesity and diabetes. Our previous report showed that Smad3 has circadian expression in mouse livers. In the current study, we focused on the heart, especially on the myocardial stromal fibroblasts because the roles of Bmal1 and Smad3 in this tissue are poorly understood. Bmal1 and Smad3 have circadian expression in mouse hearts, and their circadian expression patterns were similar. Bmal1 expression decreased in the hearts of whole-body Smad3 knockout mice, whereas Smad3 expression had little effect on heart-specific Bmal1 knockout mice. Both Smad3 knockout and heart-specific Bmal1 knockout mice showed increases in p21, S100A4, CD206 and TNF-α expression in the myocardial stromal fibroblasts and macrophage compared to control mice. We also examined Smad3, Bmal1 and Dec1 expression in human tissue from old myocardial infarctions. Expression of Smad3, Bmal1 and Dec1 decreased in the stromal fibroblasts of tissue from old myocardial infarctions compared to control cases. On the other hand, p21, S100A4 and TNF-α increased in the stromal fibroblasts of tissue from old myocardial infarctions. Furthermore, expression of Smad3, Bmal1 and Dec1 decreased in TNF-α treated-NIH3T3 cells but expression of p21 and S100A4 increased. This new evidence suggests that Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts through TNF-α.

Acidic microenvironments induce lymphangiogenesis and IL-8 production via TRPV1 activation in human lymphatic endothelial cells.

Local acidosis is one of the characteristic features of the cancer microenvironment. Many reports indicate that acidosis accelerates the proliferation and invasiveness of cancer cells. However, whether acidic conditions affect lymphatic metastasis is currently unknown. In the present study, we focused on the effects of acidosis on lymphatic endothelial cells (LECs) to assess the relationship between acidic microenvironments and lymph node metastasis. We demonstrated that normal human LECs express various acid receptors by immunohistochemistry and reverse transcriptase-polymerase chain reaction (PCR). Acidic stimulation with low pH medium induced morphological changes in LECs to a spindle shape, and significantly promoted cellular growth and tube formation. Moreover, real-time PCR revealed that acidic conditions increased the mRNA expression of interleukin (IL)-8. Acidic stimulation increased IL-8 production in LECs, whereas a selective transient receptor potential vanilloid subtype 1 (TRPV1) antagonist, 5'-iodoresiniferatoxin, decreased IL-8 production. IL-8 accelerated the proliferation of LECs, and inhibition of IL-8 diminished tube formation and cell migration. In addition, phosphorylation of nuclear factor (NF)-κB was induced by acidic conditions, and inhibition of NF-κB activation reduced acid-induced IL-8 expression. These results suggest that acidic microenvironments in tumors induce lymphangiogenesis via TRPV1 activation in LECs, which in turn may promote lymphatic metastasis.

Levels of serum-circulating angiogenic factors within 1 week prior to delivery are closely related to conditions of pregnant women with pre-eclampsia, gestational hypertension, and/or fetal growth restriction.

AIM: We aimed to investigate maternal serum angiogenic marker profiles within 1 week prior to delivery in cases of gestational hypertension (GH), pre-eclampsia (PE), and/or fetal growth restriction (FGR) with different clinical conditions. METHODS: We enrolled 165 women with singleton pregnancy. The participants were classified based on three characteristics: (i) proteinuria (GH and PE); (ii) FGR (PE with FGR [PE + FGR], PE alone, and FGR alone); and (iii) onset (early onset PE [EO PE] and late-onset PE [LO PE]). All sera were obtained within 1 week prior to delivery, and soluble fms-like tyrosine kinase 1 (sFlt-1), soluble endoglin (sEng), and placental growth factor (PlGF) were measured with enzyme-linked immunosorbent assay. RESULTS: (i) In PE, a significantly increased sFlt-1, sEng, and sFlt-1 to PlGF ratio (sFlt-1/PlGF) and significantly decreased PlGF were observed compared with GH and Term control, whereas in GH, only sFlt-1/PlGF was significantly higher than Term control. (ii) In PE + FGR, similar changes were more markedly shown compared with PE alone. The FGR alone group exhibited similar tendencies as PE, although significant differences were found in PlGF and sEng levels. (iii) In EO PE, significant changes were observed in all factors compared with LO PE or Term control, while no significant change in PlGF levels was observed between LO PE and Term control. CONCLUSION: We demonstrated that the levels of circulating angiogenic factors just before delivery are correlated with the severity of hypertensive disorders of pregnancy and FGR. Profiling these specific markers may contribute to better understanding of the clinical conditions in individual patients and their pathogenesis.

DEC1 negatively regulates AMPK activity via LKB1.

Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) is one of the clock genes that show a circadian rhythm in various tissues. AMP-activated protein kinase (AMPK) activity plays important roles in the metabolic process and in cell death induced by glucose depletion. Recent reports have shown that AMPK activity exhibited a circadian rhythm. However, little is known regarding the regulatory mechanisms involved in the circadian rhythm of AMPK activity. The aim of this study is to investigate whether there is a direct correlation between DEC1 expression and AMPK activity. DEC1 protein and AMPK activity showed a circadian rhythm in the mouse liver with different peak levels. Knocking down DEC1 expression increased AMPK activity, whereas overexpression of DEC1 decreased it. Overexpressing the DEC1 basic mutants had little effect on the AMPK activity. DEC1 bound to the E-box of the LKB1 promoter, decreased LKB1 activity and total protein levels. There was an inverse relationship between DEC1 expression and AMPK activity. Our results suggest that DEC1 negatively regulates AMPK activity via LKB1.

Trps1 deficiency inhibits the morphogenesis of secondary hair follicles via decreased Noggin expression.

A representative phenotype of patients with tricho-rhino-phalangeal syndrome (TRPS) is sparse hair. To understand the developmental defects of these patient's hair follicles, we analyzed the development of hair follicles histologically and biochemically using Trps1 deficient (KO) mice. First, we compared the numbers of primary hair follicles in wild-type (WT) and KO embryos at different developmental stages. No differences were observed in the E14.5 skins of WT and KO mice. However, at later time points, KO fetal skin failed to properly develop secondary hair follicles, and the number of secondary hair follicles present in E18.5 KO skin was approximately half compared to that of WT skin. Sonic hedgehog expression was significantly decreased in E17.5 KO skin, whereas no changes were observed in Eda/Edar expression in E14.5 or E17.5 skins. In addition, Noggin expression was significantly decreased in E14.5 and E17.5 KO skin compared to WT skin. In parallel with the suppression of Noggin expression, BMP signaling was promoted in the epidermal cells of KO skins compared to WT skins as determined by immunohistochemistry for phosphorylated Smad1/5/8. The reduced number of secondary hair follicles was restored in skin graft cultures treated with a Noggin and BMP inhibitor. Furthermore, decreased cell proliferation, and increased apoptosis in KO skin was rescued by Noggin treatment. Taken together, we conclude that hair follicle development in Trps1 KO embryos is impaired directly or indirectly by decreased Noggin expression.

The loss of Trps1 suppresses ureteric bud branching because of the activation of TGF-ß signaling.

In a previous study, we demonstrated that Trps1-deficient (KO) mice show an expanded renal interstitium compared to wild-type (WT) mice because the loss of Trps1 affects the mesenchymal-epithelial transition (MET) in the cap mesenchyme and ureteric bud (UB) branching. Although we previously elucidated the mechanism underlying the impact of Trps1 on the MET, how Trps1 is involved in UB branching remains unknown. In the present study, we unveil the molecular mechanisms by which the loss of Trps1 suppresses UB branching. When we compared gene expression patterns via DNA microarray analysis using cultured ureteric buds isolated from E11.5 kidneys of WT and KO embryos, we found aberrant expression of genes associated with the transforming growth factor (TGF)-ß/Smad3 signaling pathway in the KO UBs. Western blot and immunohistochemistry analyses showed increased levels of Rb1cc1, Arkadia1, and phosphorylated Smad3 and decreased levels of Smurf2, Smad7, and c-Ski in the KO embryonic kidneys. In addition, TUNEL staining and immunohistochemical detection of PCNA revealed that the apoptosis of UB cells was upregulated and, conversely, that cell proliferation was suppressed. Finally, we demonstrated that the suppression of UB branching in the KO UBs was restored via the exogenous addition of the Smad3 inhibitor SIS3, whereas the addition of TGF-ß1 accelerated the suppression of UB branching in organ cultures of both isolated UBs and whole embryonic kidneys. Considering these results, we conclude that UB branching is suppressed through increased activation of the TGF-ß/Smad3 signaling pathway when Trps1 is lost.

Smad3 plays an inhibitory role in phosphate-induced vascular smooth muscle cell calcification.

Arterial medial calcification is a major complication in patients with chronic kidney disease and diabetes. It has been hypothesized that a high concentration of inorganic phosphate (Pi) induces calcification in vascular smooth muscle cells (vSMCs). However, the role of transforming growth factor-ß (TGF-ß)/Smad3 signaling in Pi-induced vascular calcification remains controversial. The aim of this study was to investigate the possible involvement of Smad3 in Pi-induced vascular calcification. We compared the degree of Pi-induced vSMC calcification between vSMCs isolated from wild-type (Smad3(+/+)) and Smad3-deficient (Smad3(-/-)) mice. We found that vSMCs from Smad3(+/+) mice had less calcium (Ca) than those from Smad3(-/-) mice when they were exposed to high concentrations of Pi and Ca (Pi+Ca). The phosphorylation of Smad3 was induced in Smad3(+/+) vSMCs by exposure to Pi+Ca. The concentration of extracellular pyrophosphate (ePPi) was lower in Smad3(-/-) vSMCs than in Smad3(+/+) vSMCs and was significantly increased in Smad3(+/+) vSMCs by treatment with TGF-ß1. Also, the addition of a small amount of PPi to culture medium significantly decreased the deposition of Ca in both Smad3(+/+) and Smad3(-/-) vSMCs. Ectonucleotide phosphatase/phosphodiesterase1 (Enpp1) was decreased at the mRNA, protein, and enzymatic activity levels in Smad3(-/-) vSMCs compared with Smad3(+/+) vSMCs. A ChIP assay showed that phosphorylated Smad3 directly binds to the Enpp1 gene. Furthermore, the calcification of aortic segments was attenuated by treatment with TGF-ß1 only in Smad3(+/+) mice. Taken together, we conclude that Pi-induced vSMC calcification is suppressed by Smad3 via an increase in ePPi.

Loss of Dec1 inhibits alcohol-induced hepatic lipid accumulation and circadian rhythm disorder.

Chronic alcohol exposure increases liver damage such as lipid accumulation and hepatitis, resulting in hepatic cirrhosis. Chronic alcohol intake is known to disturb circadian rhythms in humans and animals. DEC1, a basic helix-loop-helix transcription factor, plays an important role in the circadian rhythm, inflammation, immune responses, and tumor progression. We have previously shown that Dec1 deficiency inhibits stresses such as periodontal inflammation and perivascular fibrosis of the heart. However, the significance of Dec1 deficiency in chronic alcohol consumption remains unclear. In the present study, we investigated whether the biological stress caused by chronic alcohol intake is inhibited in Dec1 knockout mice. We treated control and Dec1 knockout mice for three months by providing free access to 10% alcohol. The Dec1 knockout mice consumed more alcohol than control mice, however, we observed severe hepatic lipid accumulation and circadian rhythm disturbance in control mice. In contrast, Dec1 knockout mice exhibited little effect on these outcomes. We also investigated the expression of peroxisome proliferator-activated receptors (PPARs) and AMP-activated protein kinase (AMPK), which are involved in the regulation of fatty acid metabolism. Immunohistochemical analysis revealed increases of phosphorylation AMPK and PPARa but decreases PPARg in Dec1 knockout mice compared to that in control mice. This indicates a molecular basis for the inhibition of hepatic lipid accumulation in alcohol-treated Dec1 knockout mice. These results suggest a novel function of Dec1 in alcohol-induced hepatic lipid accumulation and circadian rhythm disorders.

Histological analysis of a Becker muscular dystrophy case, diurnal expression of dystrophin in control mice and decreased expression of dystrophin in Bmal1 knockout mice.

Becker muscular dystrophy (BMD) is a hereditary disease characterized by dystrophin deletion that consequently induces muscle weakness, cardiac hypertrophy and cardiac failure; These conditions are similar to those in Duchenne muscular dystrophy. The circadian rhythm is a physiological phenomenon that is predominantly regulated by the transcription and translation of clock genes. Bmal1 (Brain and muscle Arnt-like protein 1) is one of the core clock genes, and its deficiency disturbs the circadian rhythm, results in cardiac hypertrophy and cardiac failure. Dystrophin expression under diurnal conditions and in Bmal1 deficiency is yet to be elucidated. In this study, we analyzed the heart and lungs sampled during a BMD autopsy. Macroscopical examination revealed a large heart and dilated cardiomyopathy. Microscopical examination revealed an undulated structure, as well as the degeneration, and necrosis of myocardial cells. We also analyzed dystrophin expression in tissues obtained from human autopsies and mice. In human autopsy cases, dystrophin expression was lower in the heart with BMD compared that in the heart with non-BMD hypertrophy. In the heart and muscle of control mice, dystrophin expression was higher at ZT0 than at ZT12. The dystrophin expression was found to be lower in heart-specific Bmal1 knockout mice compared to that in the control mice. Hence, our study indicated that BMD was closely associated with cardiac hypertrophy and cardiac failure, while dystrophin had a diurnal expression pattern in control mice that was regulated by Bmal1.

MicroRNAs that target Ca(2+) transporters are involved in vascular smooth muscle cell calcification.

The role of microRNAs (miRNAs) in vascular calcification is currently unclear. To examine how miRNAs are involved in vascular smooth muscle cell (VSMC) calcification, we explored the alteration of miRNAs in VSMC calcification in vitro and in vivo. Klotho homozygous mutant mice (kl/kl) display vascular calcification and have perturbations of calcium handling. We therefore hypothesized that the calcium perturbations in VSMCs could be mediated by miRNAs. Using an miRNA array analysis, we demonstrated that miRNAs are aberrantly expressed in the aortic media of 3-week-old kl/kl mice compared with wild-type (WT) mice. The expression levels of miR-135a(*), miR-762, miR-714, and miR-712(*) in the aortic media of kl/kl mice were significantly higher than in WT mice. We used quantitative real-time reverse transcriptase polymerase chain reaction to further confirm that these miRNAs were increased in the aortic media of kl/kl mice and in cultured VSMCs treated with high phosphate and calcium. A search of the miRNA database indicated that the Ca(2+) efflux proteins NCX1, PMCA1, and NCKX4 frequently appeared as potential targets of these miRNAs. The transfection of miRNA mimics into cultured VSMCs reduced the protein levels of each potential target. Conversely, miRNA inhibitors reduced phosphate and calcium-induced VSMC calcification. Furthermore, these inhibitors decreased the intracellular Ca(2+) concentration in cultured VSMCs after treatment with phosphate and calcium. Our results suggest that increased expression of miR-135a(*), miR-762, miR-714, and miR-712(*) in VSMCs may be involved in VSMC calcification by disrupting Ca(2+) efflux proteins.

Trps1 is necessary for normal temporomandibular joint development.

Mutation of the human TRPS1 gene leads to trichorhinophalangeal syndrome (TRPS), which is characterized by an abnormal development of various organs including the craniofacial skeleton. Trps1 has recently been shown to be expressed in the jaw joints of zebrafish; however, whether Trps1 is expressed in the mammalian temporomandibular joint (TMJ), or whether it is necessary for TMJ development is unknown. We have analyzed (1) the expression pattern of Trps1 during TMJ development in mice and (2) TMJ development in Trps1 knockout animals. Trps1 is expressed in the maxillo-mandibular junction at embryonic day (E) 11.5. At E15.5, expression is restricted to the developing condylar cartilage and to the surrounding joint disc progenitor cells. In Trps1 knockout mice, the glenoid fossa of the temporal bone forms relatively normally but the condylar process is extremely small and the joint disc and cavities do not develop. The initiation of condyle formation is slightly delayed in the mutants at E14.5; however, at E18.5, the flattened chondrocyte layer is narrowed and most of the condylar chondrocytes exhibit precocious chondrocyte maturation. Expression of Runx2 and its target genes is expanded toward the condylar apex in the mutants. These observations underscore the indispensable role played by Trps1 in normal TMJ development in supporting the differentiation of disc and synoviocyte progenitor cells and in coordinating condylar chondrocyte differentiation.

Human prolyl hydroxylase expression in uterine leiomyoma during the menstrual cycle.

BACKGROUND: To investigate the role of prolyl hydroxylase (PH), a key enzyme of collagen synthesis, in human uterine leiomyoma, PH expression was determined in the normal uterine myometrium and the leiomyoma tissues during the menstrual cycle. METHODS: The tissues were obtained from 40 regularly cycling women (aged 29 to 53 yr) who were undergoing abdominal hysterectomy for symptomatic uterine leiomyoma. Immunohistochemistry for human PH with specific monoclonal antibody was used for analysis. RESULTS: Immunohistochemical staining for PH revealed intense staining of leiomyoma cells in the uterine leiomyoma throughout the menstrual cycle, as compared with the adjacent normal myometrium. In the secretory phase, weak or no immunostaining for PH was detected in the normal myometrial tissues. CONCLUSIONS: These results suggest that increased expression of PH might play an role in the physiology of uterine leiomyoma during the menstrual cycle.

Diverse roles of macrophages in atherosclerosis: from inflammatory biology to biomarker discovery.

Cardiovascular disease, a leading cause of mortality in developed countries, is mainly caused by atherosclerosis, a chronic inflammatory disease. Macrophages, which differentiate from monocytes that are recruited from the blood, account for the majority of leukocytes in atherosclerotic plaques. Apoptosis and the suppressed clearance of apoptotic macrophages (efferocytosis) are associated with vulnerable plaques that are prone to rupture, leading to thrombosis. Based on the central functions of macrophages in atherogenesis, cytokines, chemokines, enzymes, or microRNAs related to or produced by macrophages have become important clinical prognostic or diagnostic biomarkers. This paper discusses the impact of monocyte-derived macrophages in early atherogenesis and advanced disease. The role and possible future development of macrophage inflammatory biomarkers are also described.

Differential immunohistochemical expression of DEC1, CK­1ε, and CD44 in oral atypical squamous epithelium and carcinoma in situ.

Presence of nuclear atypia during histological investigation is often a cause of concern for pathologists while identifying tumor and non­tumor cells in a biopsy sample of oral mucosa. Nuclear atypia is observed in severe inflammation, ulcers and reactive changes. Therefore, additional methods, such as immunohistochemistry, may help precise diagnosis. When the atypia is suggestive of tumorous or reactive origin, the lesion is diagnosed as atypical squamous epithelium (ASE). When there is severe nuclear atypia in the mucosa, such as in disorders of nuclear polarity, large nuclei, and clear nucleolus, the lesion is diagnosed as carcinoma in situ (CIS). However, it is not easy to distinguish ASE and CIS using hematoxylin and eosin staining. The present study aimed to distinguish ASE from CIS using immunohistochemistry. A total of 32 biopsy samples of either ASE or CIS cases were selected and the level of casein kinase 1ε (CK­1ε), differentiated embryonic chondrocyte gene 1 (DEC1), proliferating cell nuclear antigen (PCNA) and CD44, which are four protein markers which have been previously linked to cancer progression, were analyzed. CK­1ε and CD44 expression was higher in CIS samples than in ASE samples. However, DEC1 expression was lower in CIS samples than in ASE samples. PCNA expression was not markedly different between the two groups. Additionally, it was found that DEC1­overexpressing cells had decreased levels of CK­1ε and CD44 compared with control cells, while CK­1ε­overexpressing cells had relatively unchanged levels of CD44, DEC1 and PCNA. These results suggested that DEC1 negatively regulates the expression of CK­1ε and CD44. Thus, DEC1, CK­1ε, and CD44 were identified as mechanistically linked and clinically relevant protein biomarkers, which could help distinguish ASE and CIS.