Epigenetic modifications precede molecular alterations and drive human hepatocarcinogenesis.

Development of primary liver cancer is a multistage process. Detailed understanding of sequential epigenetic alterations is largely missing. Here, we performed Infinium Human Methylation 450k BeadChips and RNA-Seq analyses for genome-wide methylome and transcriptome profiling of cirrhotic liver (n = 7), low- (n = 4) and high-grade (n = 9) dysplastic lesions, and early (n = 5) and progressed (n = 3) hepatocellular carcinomas (HCC) synchronously detected in 8 patients with HCC with chronic hepatitis B infection. Integrative analyses of epigenetically driven molecular changes were identified and validated in 2 independent cohorts comprising 887 HCCs. Mitochondrial DNA sequencing was further employed for clonality analyses, indicating multiclonal origin in the majority of investigated HCCs. Alterations in DNA methylation progressively increased from liver cirrhosis (CL) to dysplastic lesions and reached a maximum in early HCCs. Associated early alterations identified by Ingenuity Pathway Analysis (IPA) involved apoptosis, immune regulation, and stemness pathways, while late changes centered on cell survival, proliferation, and invasion. We further validated 23 putative epidrivers with concomitant expression changes and associated with overall survival. Functionally, Striatin 4 (STRN4) was demonstrated to be epigenetically regulated, and inhibition of STRN4 significantly suppressed tumorigenicity of HCC cell lines. Overall, application of integrative genomic analyses defines epigenetic driver alterations and provides promising targets for potentially novel therapeutic approaches.

Adding myofibroblasts to the lacrimal pump.

The lacrimal sac (LS) empties in the nasolacrimal duct to drain the tears in the inferior nasal meatus. Different studies indicated the role of the lacrimal pump in the lacrimal drainage. Although controversial, the lacrimal pump mechanism is an extrinsic one, either active, or passive. An intrinsic contractile potential of the LS was not documented previously. We thus aimed a retrospective immunohistochemical study to test the alpha-smooth muscle actin (α-SMA) and h-caldesmon expression in the LS wall. We used archived paraffin-embedded samples of LS from ten adult patients. The α-SMA + phenotype was detected in basal epithelial cells, in subepithelial ribbons of stromal cells, in vascular smooth muscle cells, as well as in pericytes. H-caldesmon was exclusively expressed in pericytes, vascular smooth muscle cells and myoepithelial cells of the subepithelial glands. The most striking feature we found in all samples was a consistent stromal network of α-SMA+/h-caldesmon- myofibroblasts. This finding supports an intrinsic scaffold useful for the lacrimal pump.

Aberrant UBR4 expressions in Hirschsprung disease patients.

BACKGROUND: Recently, pathogenic alleles within ubiquitin N-recognin domain-containing E3 ligase 4 (UBR4) gene have been shown to be associated with Hirschsprung disease (HSCR). We determined the UBR4 expressions in Indonesian HSCR patients. METHODS: We analyzed the UBR4 expressions in the colons of HSCR patient and anorectal malformation (ARM) patient as control by real-time polymerase chain reaction (qPCR). RESULTS: Thirty-seven patients with non-syndromic HSCR and eighteen controls were involved in this study. qPCR revealed that the UBR4 expression was strongly decreased (0.77-fold) in the ganglionic group of patients with HSCR compared to the control group with ARM (ΔCT 2.43 ± 0.36 vs. 2.05 ± 0.69; p = 0.009), whereas the UBR4 expression was also significantly reduced (0.79-fold) in the aganglionic group of patients with HSCR compared to the control group with ARM (ΔCT 2.39 ± 0.46 vs. 2.05 ± 0.69; p = 0.044). However, the UBR4 expression change was not associated with gender (p = 0.35 and 0.80), nor with degree of aganglionosis both in ganglionic and aganglionic colons (p = 0.72 and 0.73), respectively. CONCLUSION: Our study demonstrates that expression of UBR4 is decreased in both aganglionic and ganglionic colon of HSCR patients.

MicroRNA Expression Profiling Screen miR-3557/324-Targeted CaMK/mTOR in the Rat Striatum of Parkinson's Disease in Regular Aerobic Exercise.

This study aimed to screen the target miRNAs and to investigate the differential miR-3557/324-targeted signal mechanisms in the rats' model of Parkinson's disease (PD) with regular aerobic exercise. Rats were divided into sedentary control PD group (SED-PD, n = 18) and aerobic exercise PD group (EX-PD, n = 22). After 8 weeks of regular aerobic exercise, a 6-hydroxydopamine- (6-OHDA-) induced PD lesion model was constructed. Preregular aerobic exercises enhanced the injury resistance of rats with 6-OHDA-induced PD. The rotational behavior after injection of apomorphine hydrochloride was alleviated. Under the scanning electron microscopy, we found the neurons, axons, and villi of the striatum were clearly and tightly arranged, and neurons and axons significantly becoming larger. Tyrosine hydroxylase (TH) was increased significantly and α-synuclein protein expression was reduced in the EX-PD group compared to the SED-PD group. Screening from miRNA microarray chip, we further found upregulation of miR-3557 and downregulation of miR-324 were closely related to the calcium-modulating signaling pathway, remitting the progress of Parkinson's disease on aerobic exercise. Compared to the SED-PD group, Ca2+/calmodulin dependent protein kinase II (CaMK2α) was upregulated, but CaMKV and voltage-dependent anion-selective channel protein 1 (Vdac1) were significantly downregulated in the EX-PD group. Additionally, phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) expression were activated, and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) expression was upregulated in the EX-PD group. Conclusions: the adaptive mechanism of regular aerobic exercise delaying neurodegenerative diseases and lesions was that miR-3557/324 was activated to regulate one of its targets CaMKs signaling pathways. CaMKs, coordinated with mTOR pathway-related gene expression, improved UCH-L1 level to favor for delaying neurodegeneration or improving the pathogenesis of PD lesions.

Adducins inhibit lung cancer cell migration through mechanisms involving regulation of cell-matrix adhesion and cadherin-11 expression.

Cell migration is a critical mechanism controlling tissue morphogenesis, epithelial wound healing and tumor metastasis. Migrating cells depend on orchestrated remodeling of the plasma membrane and the underlying actin cytoskeleton, which is regulated by the spectrin-adducin-based membrane skeleton. Expression of adducins is altered during tumorigenesis, however, their involvement in metastatic dissemination of tumor cells remains poorly characterized. This study investigated the roles of α-adducin (ADD1) and γ-adducin (ADD3) in regulating migration and invasion of non-small cell lung cancer (NSCLC) cells. ADD1 was mislocalized, whereas ADD3 was markedly downregulated in NSCLC cells with the invasive mesenchymal phenotype. CRISPR/Cas9-mediated knockout of ADD1 and ADD3 in epithelial-type NSCLC and normal bronchial epithelial cells promoted their Boyden chamber migration and Matrigel invasion. Furthermore, overexpression of ADD1, but not ADD3, in mesenchymal-type NSCLC cells decreased cell migration and invasion. ADD1-overexpressing NSCLC cells demonstrated increased adhesion to the extracellular matrix (ECM), accompanied by enhanced assembly of focal adhesions and hyperphosphorylation of Src and paxillin. The increased adhesiveness and decreased motility of ADD1-overexpressing cells were reversed by siRNA-mediated knockdown of Src. By contrast, the accelerated migration of ADD1 and ADD3-depleted NSCLC cells was ECM adhesion-independent and was driven by the upregulated expression of pro-motile cadherin-11. Overall, our findings reveal a novel function of adducins as negative regulators of NSCLC cell migration and invasion, which could be essential for limiting lung cancer progression and metastasis.

17ß-estradiol upregulates striatin protein levels via Akt pathway in human umbilical vein endothelial cells.

17ß-estradiol (E2) has been shown to have beneficial effects on the cardiovascular system. We previously demonstrated that E2 increases striatin levels and inhibits migration in vascular smooth muscle cells. The objective of the present study was to investigate the effects of E2 on the regulation of striatin expression in human umbilical vein endothelial cells (HUVECs). We demonstrated that E2 increased striatin protein expression in a dose- and time-dependent manner in HUVECs. Pretreatment with ICI 182780 or the phosphatidylinositol-3 kinase inhibitor, wortmannin, abolished E2-mediated upregulation of striatin protein expression. Treatment with E2 resulted in Akt phosphorylation in a time-dependent manner. Moreover, silencing striatin significantly inhibited HUVEC migration, while striatin overexpression significantly promoted HUVEC migration. Finally, E2 enhanced HUVEC migration, which was inhibited by silencing striatin. In conclusion, our results demonstrated that E2-mediated upregulation of striatin promotes cell migration in HUVECs.

TGFß1-induced expression of caldesmon mediates epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is an important process that mediates organ development and wound healing, and in pathological contexts, it can contribute to the progression of fibrosis and cancer. During EMT, cells exhibit marked changes in cytoskeletal organization and increased expression of a variety of actin associated proteins. Here, we sought to determine the role of caldesmon in mediating EMT in response to transforming growth factor (TGF)-ß1. We find that the expression level and phosphorylation state of caldesmon increase as a function of time following induction of EMT by TGFß1 and these changes in caldesmon correlate with increased focal adhesion number and size and increased cell contractility. Knockdown and forced expression of caldesmon in epithelial cells reveals that caldesmon expression plays an important role in regulating the expression of the myofibroblast marker alpha smooth muscle actin. Results from these studies provide insight into the role of cytoskeletal associated proteins in the regulation of EMT and may suggest ways to target the cell cytoskeleton for regulating EMT processes.

Compared With Elastin Stains, h-Caldesmon and Desmin Offer Superior Detection of Vessel Invasion in Gastric, Pancreatic, and Colorectal Adenocarcinomas.

BACKGROUND: The presence of vessel invasion is considered indicative of a poor prognosis in many malignant tumors. We aimed to compare the sensitivity of elastin stains (van Gieson's and orcein methods) with 2 smooth muscle markers (h-caldesmon and desmin) in gastric, pancreatic, and colorectal adenocarcinoma specimens. MATERIALS AND METHODS: We used 27 (29.3%) gastric, 35 (38.0%) pancreatic, and 30 (32.6%) colorectal resection specimens. We applied a provisional classification of vessel invasion patterns: type A, a focus with a nearby artery unaccompanied by a vein; type T, a focus at the invasive front without an unaccompanied artery; and type X, foci that only appeared by any of the 4 stains used. RESULTS: There were 369 foci. The smooth muscle markers were more sensitive than the elastin stains, and h-caldesmon more sensitive than desmin, in all types. Among the 139 type A foci, 33 (23.7%) were positive by desmin and h-caldesmon, whereas the elastin stains were not ( P = .001). h-Caldesmon was the only positive marker in 11 (7.9%; P = .011). Among the 78 type T foci, 21 (26.9%) were positive by desmin and h-caldesmon, when both elastin stains were negative ( P = .000). In 16 (20.5%) foci, h-caldesmon was the only positive marker ( P = .002). Among 152 type X foci, 91 (59.9%) were positive by all markers, 26 (17.1%) by both desmin and h-caldesmon, and 9 (5.9%) by only the 2 elastin stains ( P = .001). CONCLUSION: We recommend these stains for suspect foci in gastric, pancreatic, and colorectal adenocarcinoma specimens. They might highlight both predictable and unpredictable foci.

Determination of dendritic spine morphology by the striatin scaffold protein STRN4 through interaction with the phosphatase PP2A.

Dendritic spines are heterogeneous and exist with various morphologies. Altered spine morphology might underlie the cognitive deficits in neurodevelopmental disorders such as autism, but how different subtypes of dendritic spines are selectively maintained along development is still poorly understood. Spine maturation requires spontaneous activity of N-methyl-d-aspartate (NMDA) receptor and local dendritic protein synthesis. STRN4 (also called zinedin) belongs to the striatin family of scaffold proteins, and some of the potential striatin-interacting proteins are encoded by autism risk genes. Although previous studies have demonstrated their localization in dendritic spines, the function of various striatin family members in the neuron remains unknown. Here, we demonstrate that Strn4 mRNA is present in neuronal dendrites, and the local expression of STRN4 protein depends on NMDA receptor activation. Notably, STRN4 is preferentially expressed in mushroom spines, and STRN4 specifically maintains mushroom spines but not thin spines and filopodia through interaction with the phosphatase PP2A. Our findings have therefore unraveled the local expression of STRN4 as a novel mechanism for the control of dendritic spine morphology.

Cell-to-cell heterogeneity of EWSR1-FLI1 activity determines proliferation/migration choices in Ewing sarcoma cells.

Ewing sarcoma is characterized by the expression of the chimeric EWSR1-FLI1 transcription factor. Proteomic analyses indicate that the decrease of EWSR1-FLI1 expression leads to major changes in effectors of the dynamics of the actin cytoskeleton and the adhesion processes with a shift from cell-to-cell to cell-matrix adhesion. These changes are associated with a dramatic increase of in vivo cell migration and invasion potential. Importantly, EWSR1-FLI1 expression, evaluated by single-cell RT-ddPCR/immunofluorescence analyses, and activity, assessed by expression of EWSR1-FLI1 downstream targets, are heterogeneous in cell lines and in tumours and can fluctuate along time in a fully reversible process between EWSR1-FLI1high states, characterized by highly active cell proliferation, and EWSR1-FLI1low states where cells have a strong propensity to migrate, invade and metastasize. This new model of phenotypic plasticity proposes that the dynamic fluctuation of the expression level of a dominant oncogene is an intrinsic characteristic of its oncogenic potential.

Early hepatocellular carcinoma with high-grade atypia in small vaguely nodular lesions.

Multistep hepatocarcinogenesis progresses from dysplastic nodules to early hepatocellular carcinoma (eHCC) and to advanced HCC. The aim of the present study was to investigate the detailed histopathological features of eHCC. We investigated 66 small vaguely nodular lesions resected from 40 patients. The degree of cellular and structural atypia and stromal invasion were assessed. The immunohistochemical expression of HCC-related markers adenylate cyclase-associated protein 2 (CAP2), heat shock protein 70 (HSP70), Bmi-1, CD34 and h-caldesmon were evaluated. Of the 66 nodules, 10 were diagnosed as low-grade dysplastic nodules (LGDN), 10 as high-grade dysplastic nodules (HGDN) and 46 as eHCC. Among the 46 eHCC, 18 nodules (39.1%) showed marked stromal invasion and/or the presence of the scirrhous component and were subclassified as high-grade eHCC (HGeHCC). The remaining 28 eHCC, which lacked these features, were subclassified as low-grade eHCC (LGeHCC) and were examined further. HGeHCC showed high levels of cellular and structural atypia and large tumor size. The immunohistochemical expression of CAP2 and the area of sinusoidal vascularization showed increases from LGDN to HGeHCC. The density of arterial tumor vessels was high in HGeHCC compared with other nodule types. Cluster analysis of these parameters subclassified 65 nodules into HGeHCC-dominant, LGeHCC and HGDN-dominant, and LGDN-dominant groups. These results indicate the increased malignant potential of HGeHCC and suggest that it is already a transitional stage to advanced HCC. We consider that our grading classification system may be valuable for considering treatment strategies for eHCC around 2 cm in diameter.

Genome-wide analysis of the IQD gene family in maize.

IQD gene family plays important roles in plant developmental processes and stress responses. To date, no systematic characterization of this gene family has been carried out in maize. In this study, 26 IQD genes, from ZmIQD1 to ZmIQD26, were identified using Blast search tools. The phylogenetic analysis showed these genes were divided into four subfamilies (IQD I-IV) and members within the same subfamily shared conserved exon/intron distribution and motif composition. The 26 ZmIQD genes are distributed unevenly on 8 of the 10 chromosomes, with 9 segmental duplication events, suggesting that the expansion of IQDs in maize was due to the segmental duplication. The analysis of Ka/Ks ratios showed that the duplicated ZmIQDs had primarily undergone strong purifying selection. In addition, the 26 ZmIQDs displayed different expression patterns at different developmental stages of maize based on transcriptome analysis. Further, quantitative real-time PCR analysis showed that all 26 ZmIQD genes were responsive to drought treatment, suggesting their crucial roles in drought stress response. Yeast two-hybrid assay proved that ZmIQD2 and ZmIQD15 can interact with ZmCaM2 and IQ or I in IQ motif is required for ZmIQD15 to combine with CaM2. Our results present a comprehensive overview of the maize IQD gene family and lay an important foundation for further analysis aimed at uncovering the biological functions of ZmIQDs in growth and development.

Genome-wide analysis of the oxyntic proliferative isthmus zone reveals ASPM as a possible gastric stem/progenitor cell marker over-expressed in cancer.

The oxyntic proliferative isthmus zone contains the main stem/progenitor cells that provide for physiological renewal of the distinct mature cell lineages in the oxyntic epithelium of the stomach. These cells are also proposed to be the potential cells-of-origin of gastric cancer, although little is known about their molecular characteristics and specific biological markers are lacking. In this study, we developed a method for serial section-navigated laser microdissection to isolate cells from the proliferative isthmus zone of rat gastric oxyntic mucosa for genome-wide microarray gene expression analysis. Enrichment analysis showed a distinct gene expression profile for the isthmus zone, with genes regulating intracellular processes such as the cell cycle and ribosomal activity. The profile was also related to stem cell transcriptional networks and stomach neoplasia. Genes expressed uniquely in the isthmus zone were associated with E2F transcription factor 1 (E2F1), which participates in the self-renewal of stem cells and in gastric carcinogenesis. One of the unique genes was Aspm [Asp (abnormal spindle) homologue, microcephaly-associated (Drosophila)]. Here we show ASPM in single scattered epithelial cells located in the proliferative isthmus zone of rat, mouse and human oxyntic mucosa, which do not seem to be actively dividing. The ASPM-expressing cells are mainly mature cell marker-deficient, except for a limited overlap with cells with neuroendocrine and tuft cell features. Further, both ASPM and E2F1 were expressed in human gastric cancer cell lines and increased and correlated in human gastric adenocarcinomas compared to non-tumour mucosa, as shown by expression profile analyses and immunohistochemistry. The association between ASPM and the transcription factor E2F1 in gastric tissue is relevant, due to their common involvement in crucial cell fate-regulatory mechanisms. Our results thus introduce ASPM as a novel possible oxyntic stem/progenitor cell marker that may be involved in both normal gastric physiology and gastric carcinogenesis.

Immaturity of smooth muscle cells in the neointima is associated with acute coronary syndrome.

BACKGROUND: Acute coronary syndrome (ACS) is mostly caused by ruptured plaques. The characteristics of rupture-prone vulnerable plaques include thin fibrous cap, large lipid core, and lower number of smooth muscle cells. Smooth muscle cells appearing in neointimal plaques are currently thought to have a uniformly synthetic phenotype, and their sub-classification has not been performed by h-caldesmon, which is supposed to be expressed in vascular smooth muscle cells that are beyond intermediately differentiated. METHODS: Stenotic coronary arteries were obtained from autopsy material of 51 adults. Cases were divided into three groups: those who died from ACS, those with a past history of ACS but died from other causes, and those without ACS history. Histological data including fibrous cap and lipid core were measured in each specimen. Immunohistochemistry for alpha-smooth muscle actin (α-SMA), h-caldesmon, and smoothelin was performed. The ratio of h-caldesmon(+) cells to α-SMA(+) cells was counted in the neointima. RESULTS: The positivity ratio of neointimal h-caldesmon decreased in a step-wise manner from cases without history of ACS through cases with past history of ACS to cases with ACS with statistical significance (P<.001). The correlation between h-caldesmon expression and progression of ACS among the different groups was more prominent than the differences in the extent of fibrous cap and lipid core. Smoothelin(+) cells were rarely observed in the neointima. CONCLUSIONS: Decreased positivity of h-caldesmon in neointimal smooth muscle cells is indicative of a more immature phenotype, thus may be associated with plaque vulnerability that will promote ACS.

Variants in striatin gene are associated with salt-sensitive blood pressure in mice and humans.

Striatin is a novel protein that interacts with steroid receptors and modifies rapid, nongenomic activity in vitro. We tested the hypothesis that striatin would in turn affect mineralocorticoid receptor function and consequently sodium, water, and blood pressure homeostasis in an animal model. We evaluated salt sensitivity of blood pressure in novel striatin heterozygote knockout mice. Compared with wild type, striatin heterozygote exhibited a significant increase in blood pressure when sodium intake was increased from restricted (0.03%) to liberal (1.6%) sodium. Furthermore, renal expression of mineralocorticoid receptor and its genomic downstream targets serum/glucocorticoid-regulated kinase 1, and epithelial sodium channel was increased in striatin heterozygote versus wild-type mice on liberal sodium intake while the pAkt/Akt ratio, readout of mineralocorticoid receptor's rapid, nongenomic pathway, was reduced. To determine the potential clinical relevance of these findings, we tested the association between single nucleotide polymorphic variants of striatin gene and salt sensitivity of blood pressure in 366 white hypertensive subjects. HapMap-derived tagging single nucleotide polymorphisms identified an association of rs2540923 with salt sensitivity of blood pressure (odds ratio, 6.25; 95% confidence interval, 1.7-20; P=0.01). These data provide the first in vivo evidence in humans and rodents that associates striatin with markers of mineralocorticoid receptor activity. The data also support the hypothesis that the rapid, nongenomic mineralocorticoid receptor pathway (mediated via striatin) has a role in modulating the interaction between salt intake and blood pressure.

SG2NA recruits DJ-1 and Akt into the mitochondria and membrane to protect cells from oxidative damage.

SG2NA is a WD-40 repeat protein with multiple protein-protein interaction domains of unknown functions. We demonstrate that it associates with the antioxidant protein DJ-1 and the survival kinase Akt. The C-terminal WD-40 repeat domain of SG2NA is required for its interaction with Akt, while DJ-1 binds it further upstream. No interaction between DJ-1 and Akt occurs in the absence of SG2NA. SG2NA, DJ-1, and Akt colocalize in mitochondria and plasma membrane. Their association is enhanced by increasing levels of reactive oxygen species up to a threshold level but falters thereafter with further increase in oxidants. Mutants of DJ-1 found in patients with familial parkinsonism are not recruited by SG2NA, suggesting its role in neuroprotection. Cells depleted of SG2NA are susceptible, while those overexpressing it are resistant to apoptosis induced by oxidative stress. Our study thus unravels a novel pathway of recruitment of Akt and DJ-1 that provides protection against oxidative stress, especially in neurons.

3'-UTR poly(T/U) tract deletions and altered expression of EWSR1 are a hallmark of mismatch repair-deficient cancers.

The genome-wide accumulation of DNA replication errors known as microsatellite instability (MSI) is the hallmark lesion of DNA mismatch repair (MMR)-deficient cancers. Although testing for MSI is widely used to guide clinical management, the contribution of MSI at distinct genic loci to the phenotype remains largely unexplored. Here, we report that a mononucleotide (T/U)16 tract located in the 3' untranslated region (3'-UTR) of the Ewing sarcoma breakpoint region 1 (EWSR1) gene is a novel MSI target locus that shows perfect sensitivity and specificity in detecting mismatch repair-deficient cancers in two independent populations. We further found a striking relocalization of the EWSR1 protein from nucleus to cytoplasm in MMR-deficient cancers and that the nonprotein-coding MSI target locus itself has a modulatory effect on EWSR1 gene expression through alternative 3' end processing of the EWSR1 gene. Our results point to a MSI target gene-specific effect in MMR-deficient cancers.

A study of α5 chain of collagen IV, caldesmon, placental alkaline phosphatase and smoothelin as immunohistochemical markers of gastrointestinal smooth muscle neoplasms.

AIMS: The histological distinction between gastrointestinal smooth muscle neoplasms (SMNs) and their differential diagnoses, especially gastrointestinal stromal tumours (GISTs), has important clinical management implications. This study aimed to investigate preliminary data suggesting that loss of the α5 chain of collagen IV (α5(IV)), placental acid phosphatase (PLAP) expression and smoothelin expression can be used diagnostically as markers of gastrointestinal SMNs. To aid this investigation, these potential markers were directly compared against caldesmon. METHODS: 31 SMNs and 111 potential differential diagnoses (16 different neoplasm types) were immunostained for caldesmon, PLAP and smoothelin. RESULTS: A pilot study indicated that loss of α5(IV) positivity was neither a specific nor sensitive marker of SMN. Caldesmon, PLAP and smoothelin were expressed by all 31 SMNs though leiomyosarcomas showed some loss of staining proportion and/or intensity. Caldesmon positivity was commonly shown by GISTs, glomus tumours and angiomyolipomas. Cytoplasmic smoothelin positivity was commonly shown by glomus tumours and angiomyolipomas, whereas PLAP positivity was shown by one desmoplastic small round cell tumour studied and only infrequently shown by angiomyolipomas. Nuclear smoothelin positivity was seen among four leiomyosarcomas but also a wide range of non-SMNs. CONCLUSIONS: α5(IV) immunohistochemistry with the A7 antibody is not a diagnostically useful marker of gastrointestinal SMNs. Both PLAP and smoothelin (cytoplasmic expression only) are as sensitive as but are more specific than caldesmon as such a marker. Further, PLAP and smoothelin immunostainings are technically reliable and can be reproducibly assessed.

Sox4-mediated caldesmon expression facilitates differentiation of skeletal myoblasts.

Caldesmon (CaD), which was originally identified as an actin-regulatory protein, is involved in the regulation of diverse actin-related signaling processes, including cell migration and proliferation, in various cells. The cellular function of CaD has been studied primarily in the smooth muscle system; nothing is known about its function in skeletal muscle differentiation. In this study, we found that the expression of CaD gradually increased as differentiation of C2C12 myoblasts progressed. Silencing of CaD inhibited cell spreading and migration, resulting in a decrease in myoblast differentiation. Promoter analysis of the caldesmon gene (Cald1) and gel mobility shift assays identified Sox4 as a major trans-acting factor for the regulation of Cald1 expression during myoblast differentiation. Silencing of Sox4 decreased not only CaD protein synthesis but also myoblast fusion in C2C12 cells and myofibril formation in mouse embryonic muscle. Overexpression of CaD in Sox4-silenced C2C12 cells rescued the differentiation process. These results clearly demonstrate that CaD, regulated by Sox4 transcriptional activity, contributes to skeletal muscle differentiation.