High mobility group AT-hook 2 regulates osteoblast differentiation and facial bone development.

The mutation and deletion of high mobility group AT-hook 2 (Hmga2) gene exhibit skeletal malformation, but almost nothing is known about the mechanism. This study examined morphological anomaly of facial bone in Hmga2-/- mice and osteoblast differentiation of pre-osteoblast MC3T3-E1 cells with Hmga2 gene knockout (A2KO). Hmga2-/- mice showed the size reduction of anterior frontal part of facial bones. Hmga2 protein and mRNA were expressed in mesenchymal cells at ossification area of nasal bone. A2KO cells differentiation into osteoblasts after reaching the proliferation plateau was strongly suppressed by alizarin red and alkaline phosphatase staining analyses. Expression of osteoblast-related genes, especially Osterix, was down-regulated in A2KO cells. These results demonstrate a close association of Hmga2 with osteoblast differentiation of mesenchymal cells and bone growth. Although future studies are needed, the present study suggests an involvement of Hmga2 in osteoblast-genesis and bone growth.

NF-κB subunit RELA suppression of mucosa-associated lymphoid tissue lymphoma translocation protein 1 expression in oral carcinoma cells.

Loss of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) expression closely associates with increased aggressive behaviors of oral carcinoma cells. It emphasizes that a mechanism to suppress the expression is an important subject for understanding carcinoma progression pathway. However, nothing is known at present. This study conducted on transcriptional regulation of the gene down-regulation. Reporter assays showed the presence of the silencer region between +402 and +501 region of MALT1 gene in oral carcinoma cells. It encoded a binding site of nuclear factor-κB subunit, RELA. RELA binding to the site was confirmed by the chromatin immunoprecipitation analyses, and deletion and mutations of the site significantly decreased the RELA binding. Short interfering RNAs for RELA up-regulated reporter gene and endogenous MALT1 protein expressions, and deletion and mutations of RELA binding site increased reporter gene expression. These results demonstrated RELA-binding to the site suppresses MALT1 expression that may facilitate oral carcinoma progression.

Domain structures of mucosa-associated lymphoid tissue lymphoma translocation 1 protein for nuclear localization in oral carcinoma cells and the proliferation inhibition.

Mucosa-associated lymphoid tissue lymphoma translocation 1 protein (MALT1) consisting of death domain, Ig-like domains and caspase-like domain is expressed in nucleus of oral carcinoma cells, and loss of the expression closely associates with disease progression and stimulates proliferation of the cells. However, nothing is known about the molecular backgrounds. In this study, eight constructs with different domain constitution of human MALT1 and six constructs were transiently and stably transfected into oral carcinoma cell lines, respectively. The immunoblot analysis showed that constructs containing caspase-like domain was expressed in nucleus and the domain-deleted constructs in cytoplasm. Immunocytochemistry of stably transfected HSC2 oral carcinoma cells confirmed the caspase-like domain-dependent nuclear localization. Involvement of domains in proliferation of stably transfected HSC2 cells was quantified by the real-time and conventional colorimetric assays. In contrast to suppression of the proliferation by full-length wild-type MALT1, any domain-deleted constructs enhanced the proliferation. Death domain construct without caspase-like domain suppressed the proliferation when it was localized in nucleus by ligating with the nuclear localization signal. These results demonstrate that nuclear localization of MALT1 in oral carcinoma cells depends on the presence of caspase-like domain and that death domain nuclear entity is responsible for MALT1 inhibition of oral carcinoma cell proliferation. Nuclear localization of death domain led by caspase-like domain may suppress oral carcinoma progression.

Minimal essential region for krüppel-like factor 5 expression and the regulation by specificity protein 3-GC box binding.

Krüppel-like factor 5 (KLF5) transcriptionally controls the proliferation-differentiation balance of epithelium and is overexpressed in carcinomas. Although genomic region modifying KLF5 expression is widespread in different types of cells, the region that commonly regulates basal expression of the genes across cell-types is uncertain. In this study we determined the minimal essential region for the expression and its regulatory transcription factors using oral carcinoma cells. A reporter assay defined a 186bp region downstream of the transcription start site and a cluster of six GC boxes (GC1-GC6) as the minimal essential region. Mutation in the GC1 or GC6 regions but not other GC boxes significantly decreased the reporter expression. The decrease by the GC1 mutation was reproduced in the 2kbp full-length promoter, but not by the GC6 mutation. Additionally, specificity proteins (Sp) that can be expressed in epithelial cells and bind GC box, Sp3 co-localized with KLF5 in oral epithelium and carcinomas and chromatin immunoprecipitation analyses showed Sp3 as the prime GC1-binding protein. Inhibition of Sp-GC box binding by mithramycin A and knockdown of Sp3 by the short interfering RNA decreased expression of the reporter gene and endogenous KLF5. These data demonstrate that a 186bp region is the minimal essential region and that Sp3-GC1 binding is essential to the basal expression of KLF5.

Krüppel-like factor 4 expression in oral carcinoma cells and hypermethylation at the gene promoter.

BACKGROUND: Krüppel-like factor 4 (KLF4) is a transcription factor regulating proliferation-differentiation balance of epithelium, and down-regulated in less-differentiated and advanced oral carcinomas. Although the expression is inactivated by the promoter hypermethylation in malignant tumor cells, it remains unknown in oral carcinoma cells. METHODS: Genomic DNA isolated from nine different oral carcinoma cell lines and a normal keratinocyte line was treated with sodium bisulfite, and methylation at KLF4 gene promoter was determined by PCR direct-sequence analysis. KLF4 expression in cells cultured with or without demethylation reagent was monitored by quantitative real-time PCR and immunoblot. RESULTS: A 237-bp promoter region spanning - 718 and - 482 of KLF4 gene was hypermethylated in oral carcinoma cells that express KLF4 at a low level, but the methylation was infrequent in cells expressing KLF4 high amount. The downstream region from - 481 to +192 was not methylated in any cell lines. Demethylation treatment of cells up-regulated the expression at mRNA and protein levels. CONCLUSION: This study demonstrated that hypermethylation at a narrow range of the promoter region down-regulates KLF4 expression, and suggests that the loss of expression by the hypermethylation contributes to oral carcinoma progression.

Krüppel-like factors 4 and 5 expression and their involvement in differentiation of oral carcinomas.

Proliferation-differentiation balance of epithelial cells is regulated by Krüppel-like factors (KLF) 4 and 5, and the unbalanced expression relates to carcinoma progression. However, little is known about the expression and role in oral carcinomas. This study examined expression of KLF4 and KLF 5 in the carcinomas by immunohistochemistry (n = 67) and the involvement in proliferation and differentiation of carcinoma cells. KLF4 was detected in keratinizing carcinoma cells and KLF5 in non-keratinizing cells. KLF4 staining declined in the patient with lymph node metastasis (P < 0.05) and in parallel with the histological dedifferentiation (P = 0.09). Exogenous overexpression of KLF4 arranged cells in a cobble-like structure with desmosomes and KLF5 elongated cells like fibroblasts without desmosomes. KLF4 suppressed fibronectin expression, and KLF5 down-regulated and degraded E-cadherin. The proliferation was not affected by KLFs. Thus, down-regulation of KLF4 and up-regulation of KLF5 may stimulate oral carcinoma progression through the dedifferentiation of carcinoma cells.

Heterogeneous tumor stromal microenvironments of oral squamous cell carcinoma cells in tongue and nodal metastatic lesions in a xenograft mouse model.

BACKGROUND: Oral squamous cell carcinoma exhibits a poor prognosis, caused by aggressive progression and early-stage metastasis to cervical lymph nodes. Here, we developed a xenograft mouse model to explore the heterogeneity of the tumor microenvironment that may govern local invasion and nodal metastasis of tumor cells. METHODS: We transplanted five oral carcinoma cell lines into the tongues of nude mice and determined tongue tumor growth and micrometastatic dissemination by serially sectioning the tongue and lymph node lesions in combination with immunohistochemistry and computer-assisted image analysis. Our morphometric analysis enabled a quantitative assessment of blood and lymphatic endothelial densities in the intratumoral and host stromal regions. RESULTS: All cell lines tested were tumorigenic in mouse tongue. The metastatic lesion-derived carcinoma cell lines (OSC19, OSC20, and HSC2) yielded a 100% nodal metastasis rate, whereas the primary tumor-derived cell lines (KOSC2 and HO-1-u-1) showed <40% metastatic potential. Immunohistochemistry showed that the individual cell lines gave rise to heterogeneous tumor architecture and phenotypes and that their micrometastatic lesions assimilated the immunophenotypic properties of the corresponding tongue tumors. Notably, OSC19 and OSC20 cells shared similar aggressive tumorigenicity in both the tongue and lymph node environments but displayed markedly diverse immunophenotypes and gene expression profiles. CONCLUSIONS: Our model facilitated comparing the tumor microenvironments in tongue and lymph node lesions. The results support that tumorigenicity and tumor architecture in the host tongue environment depend on the origin and properties of the carcinoma cell lines and that metastatic progression may take place through heterogeneous tumor-host interactions.

Differential expression of fatty acid-binding proteins and pathological implications in the progression of tongue carcinoma.

Tongue carcinomas are common malignancies of the oral cavity. Understanding the molecular mechanisms behind the disease progression is a prerequisite for improving patient prognosis. Fatty acid-binding proteins (FABPs) are cytoplasmic lipid chaperones that affect cellular organization and energy production. Although their aberrant expression is involved in carcinoma progression, its role in the pathology of tongue carcinomas remains unclear. In the present study, the immunohistochemical expression of FABP4 and FABP5 in tongue carcinomas (n=58) and its involvement in the clinicopathological parameters were examined. Normal tongue epithelial cells expressed FABP5, an epidermal-type FABP, but not FABP4, an adipocyte-type FABP. The cytoplasmic staining of FABP5 was increased in carcinomas with advanced T-stage (P<0.05) and clinical stage (P<0.05). Ectopic expression of FABP4 was detected in almost all carcinomas, although its role in disease progression remains undetermined. Upregulation of FABP5 in the wounded skin of genetically normal mice indicated that microenvironmental tissue factors induce FABP5 expression. The results of the present study demonstrated the aberrant expression of FABP4 and FABP5 in tongue carcinomas and suggested the involvement of FABP5 in disease progression.

Proteomic identification of keratin alterations with enhanced proliferation of oral carcinoma cells by loss of mucosa-associated lymphoid tissue 1 expression.

Progression of oral carcinomas associates with aberrant activation and inactivation of molecules that work in established or unknown pathways. Although mucosa­associated lymphoid tissue 1 (MALT1) expressed in normal oral epithelium is inactivated in the aggressive subset of carcinomas with worse prognosis, phenotypic changes of carcinoma cells upon the loss of expression is unknown. We performed a proteomic analysis to identify MALT1­regulated proteins in oral carcinoma cells. Four different keratins were included in the ten most abundantly changed proteins. K8/18 were upregulated in MALT1 stably­expressing carcinoma cells and K5/14 in MALT1­marginal control cells. K8/18 upregulation and K5/14 downregulation were MALT1 dose­dependent and observed in a series of oral carcinoma cells. MALT1 suppressed cell proliferation (0.52-fold, P<0.01) and its dominant-negative form stimulated it (1.33-fold, P<0.01). The decreased proliferation associated with reduction of cyclin D1, which was recovered by the short interfering RNA against MALT1. Taken together, loss of MALT1 expression alters keratin expression and enhances proliferation of carcinoma cells, and may progress oral carcinomas into the advanced state.

Epigenetic loss of mucosa-associated lymphoid tissue 1 expression in patients with oral carcinomas.

Mucosa-associated lymphoid tissue 1 (MALT1), which is located in a genomic region that encodes unknown tumor suppressor gene(s), activates nuclear factor-kappaB in lymphocyte lineages. However, its expression and role in the pathology of malignant tumors of epithelial origin is not known. In the present study, we examined MALT1 expression and its implications for the pathology of oral carcinomas. Immunostaining localized MALT1 in the nucleus of normal oral epithelial cells, but the expression was absent in 45.0% of carcinomas (49 of 109 cases) especially at the invasive front. The loss of expression was correlated with tumor recurrence (P = 0.007) and poor patient survival (P < 0.001), and it was an independent prognostic determinant (P < 0.001). MALT1-negative carcinomas exhibited microsatellite instability at the MALT1 locus and a specific cytosine methylation positioned at -256 from the gene, and the expression was recovered by demethylation treatment. In contrast to lymphocyte lineages, carcinoma cells showed MALT1 located at the nucleus independent of its domain structures, and its loss of expression induced the epithelial-mesenchymal transition. These results show that MALT1 is expressed in the nucleus of oral epithelial cells and that its expression is epigenetically inactivated during tumor progression, suggesting that the detection of MALT1 expression is a useful predictive and prognostic determinant in the clinical management of oral carcinomas.

Identification of GATA3 binding sites in Jurkat cells.

Determining binding sites of transcription factors is important for understanding the transcriptional control of target genes. Although a transcription factor GATA3 plays a pivotal role in Th2 lymphocyte development, its physiological role is not clearly defined because the target genes remain largely unknown. In this study, we modified chromatin immunoprecipitation (ChIP), and isolated 121 GATA3 binding sites and 83 different annotated target genes. Re-ChIP analysis using anti-GATA3 and anti-RNA polymerase II mAbs and chromosome conformation capture assay demonstrate that GATA3-bound fragments interact with basal transcriptional units of target genes. GATA3 regulation of target genes under the control of binding fragments was confirmed by reporter assay and quantification of target gene mRNA expression in the presence of GATA inhibitor or short interfering RNA against GATA3. These data demonstrate that GATA3 binds to regulatory elements and controls target gene expression through physical interaction with core promoter regions.

The role of WNT in rheumatoid arthritis and its therapeutic implication.

Rheumatoid arthritis (RA) is a systematic inflammatory and intractable disease, which progressively affects multiple joints. Recent findings strongly suggest a key role of WNT signaling in the disease initiation and progression. In this review, we discuss the role and possibility of treatment by targeting WNT signaling.

Epigenetic inactivation of IkappaB Kinase-alpha in oral carcinomas and tumor progression.

PURPOSE: The loss of epithelial phenotypes in the process of carcinoma progression correlates with clinical outcome, and genetic/epigenetic changes accumulate aggressive clones toward uncurable disease. IkappaB kinase-alpha (IKKalpha) has a decisive role in the development of the skin and establishes keratinocyte phenotypes. We assessed clinical implications of IKKalpha expression in oral carcinomas and epigenetic aberrations for the loss of expression. EXPERIMENTAL DESIGN: We examined IKKalpha expression in oral carcinomas by immunostaining (n = 64) and genetic instability by microsatellite PCR (n = 46). Promoter methylation status was analyzed by bisulfite-modified sequence (n = 11). RESULTS: IKKalpha was expressed in the nucleus of basal cells of normal oral epithelium, but not or marginally detected in 32.8% of carcinomas. The immunoreactivity was significantly decreased in less differentiated carcinomas (P < 0.05) and correlated to long-term survival of patients (P < 0.01) with an independent prognostic value (P < 0.05). Although allelic/biallelic loss of the gene was limited to four cases, we detected microsatellite instability in 63.0% cases in which the immunoreactivities were decreased and the promoter was hypermethylated. CONCLUSION: These results showed that oral carcinomas exhibiting genetic instability and promoter hypermethylation down-regulate expression of IKK and suggest that the epigenetic loss of the expression closely associates with disease progression toward unfavorable prognosis.

Epigenetic inactivation of E-cadherin by promoter hypermethylation in oral carcinoma cells.

The loss of E-cadherin expression by epigenetic aberrations, including promoter hypermethylation and transcription repressor binding, plays a key role in the initiation of the epithelial-mesenchymal transition, which leads to the progression of oral squamous cell carcinomas. However, mutual actions and roles of the epigenetic pathways remain to be elucidated. In this study, we determined the methylation status of cytosine within CpG islands of the E-cadherin promoter region in relation to the expression level of SIP1, a major E-cadherin repressor in oral carcinoma cells. Methylation-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism analyses showed that the expression of E-cadherin was downregulated in parallel with promoter hypermethylation. The use of a bisulfite-modified sequence further validated that methylation was observed in 22.6 +/- 38.7% (mean +/- 1 SD) of cytosines in carcinoma cells negligibly expressing E-cadherin, in contrast to 7.5 +/- 1.8% in E-cadherin-expressing cells. Treatment with a demethylating reagent, 5-azacytidine, induced upregulation of E-cadherin in some E-cadherin-expressing carcinoma cell lines but not in others. The finding that the unresponsive cell lines retained high expression of SIP1 supports the repressive effect of SIP1 on E-cadherin expression regardless of promoter hypermethylation. Collectively, the overall results suggest the dynamic but differential regulation of E-cadherin by epigenetic aberrations in the pathology of oral carcinomas.

Expression of SIP1 in oral squamous cell carcinomas: implications for E-cadherin expression and tumor progression.

Loss of E-cadherin expression allows carcinoma cells to liberate from the primary site and enhances invasion and metastasis. The genetic aberration of E-cadherin is a rare event in sporadic carcinomas, and transcription repressors are considered to take a central role in E-cadherin loss. However, expression of E-cadherin repressors is largely dependent on tissue and cell type. To identify the repressor expressed in oral squamous carcinomas, we compared the expression levels of E-cadherin and repressors by real-time RT-PCR. Among the repressors including SNAIL, SLUG, SIP1, E12 and E47, SIP1 was inversely correlated to E-cadherin (P < 0.05). Chromatin immunoprecipitation showed that SIP1 specifically bound to the E-cadherin promoter region. SIP1 expression was immuno-histochemically detected in 27.7% of 47 oral carcinomas, and SIP1-positive carcinomas did not express E-cadherin (P < 0.01). Thirteen patients with SIP1 staining showed a lower disease-specific survival rate (P < 0.05). Multivariate risk factor analysis demonstrated that SIP1 expression was an independent prognostic value for disease-specific overall survival (P < 0.05). These results suggest that SIP1 contributes to the loss of E-cadherin expression and that detection of SIP1 expression is a predictive and prognostic tool in clinical management of oral carcinomas.

Differential expression and localization of WNTs in an animal model of skin wound healing.

Wound healing is a dynamic process, and a variety of growth factors have a significant impact on the process. Although the WNT family has a multitude of effects on the state of various physiological pathways, the expression and role of WNT in wounded tissue have remained an enigma. The aim of this study was to assess the expression and localization of WNTs in a murine model of wound healing. RNA isolated from full-thickness cutaneous wounds from day 1 to day 21 postwounding were subjected to reverse transcription-polymerase chain reaction, and expression of WNT3, 4, 5a, and 10b were observed. Immunohistochemistry localized WNT10b to regenerating epithelial cells on day 1 and 3, and WNT4 on day 3 and 5. WNT4 also reacted with fibroblast-like cells beneath the epithelium. The cytoplasmic staining of beta-catenin, a WNT signaling molecule, in the epithelial cells indicates an activation of the WNT signaling pathway. Among target genes downstream of the pathway, matrix metalloproteinases (MMPs) degrade and remodel the extracellular matrix during wound healing. Gelatin zymography showed that MMP9 was expressed from day 1 to day 5. MMP-2 was continuously expressed, but maximally up-regulated at day 5. Activation of MMP-2 coincided with expression of membrane-type 1 MMP, suggesting an involvement of WNTs in this proteolytic cascade. Therefore, WNTs may contribute to the process of wound healing in a spatiotemporal manner.