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.

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.

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 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.

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.

Overexpression of caldesmon is associated with lymph node metastasis and poorer prognosis in patients with oral cavity squamous cell carcinoma.

BACKGROUND: A previous comparative tissue proteomics study by the authors of the current study led to the identification of caldesmon (CaD) as one of the proteins associated with cervical metastasis of oral cavity squamous cell carcinoma (OSCC). In the current investigation, the authors focused on the potential functions of CaD in patients with OSCC. METHODS: CaD expression was examined in tissue samples from 155 patients using immunohistochemical analysis. The expression of CaD variants was determined by Western blot analysis and reverse transcriptase-polymerase chain reaction. In addition, the specific effects of CaD gene overexpression and silence were determined in OSCC cell lines. RESULTS: CaD expression was found to be significantly higher in tumor cells from metastatic lymph nodes compared with primary tumor cells, and was nearly absent in normal oral epithelia. Higher CaD expression was found to be correlated with positive N classification, poor differentiation, perineural invasion, and tumor depth (P = .001, P = .029, P = .001, and P = .031, respectively). In survival analyses, OSCC patients with higher CaD expression were found to have poorer prognosis with regard to disease-specific survival and disease-free survival (P = .003 and P = .014, respectively). Multivariate analyses further indicated that higher CaD expression was an independent predictor of disease-specific survival (P = .043). Serum CaD levels were found to be significantly higher in patients with OSCC, but this finding was not associated with clinicopathological manifestations. Data obtained from in vitro suppression, rescue, and overexpression of CaD in OEC-M1 cells indicated that CaD promotes migration and invasive processes in OSCC cells. CONCLUSIONS: The findings of the current study collectively suggest that the low-molecular-weight CaD expression in OSCC tumors is associated with tumor metastasis and patient survival.

Activation of the mineralocorticoid receptor increases striatin levels.

BACKGROUND: Aldosterone (ALDO), a critical regulator of sodium homeostasis, mediates its effects via activation of the mineralocorticoid receptor (MR) through mechanisms that are not entirely clear. Striatin, a membrane associated protein, interacts with estrogen receptors in endothelial cells. METHODS: We studied the effects of MR activation in vitro and in vivo on striatin levels in vascular tissue. RESULTS: We observed that dietary sodium restriction was associated with increased striatin levels in mouse heart and aorta and that striatin and MR are present in the human endothelial cell line, (EA.hy926), and in mouse aortic endothelial cells (MAEC). Further, we show that MR co-precipitates with striatin in vascular tissue. Incubation of EA.hy926 cells with ALDO (10(-8) mol/l for 5-24 h) increases striatin protein and mRNA expression, an effect that was inhibited by canrenoic acid, an MR antagonist. Consistent with these observations, incubation of MAEC with ALDO increased striatin levels that were likewise blocked by canrenoic acid. To test the in vivo relevance of these findings, we studied two previously described mouse models of increased ALDO levels. Intraperitoneal ALDO administration augmented the abundance of striatin protein in mouse heart. We also observed that in a murine model of chronic ALDO-mediated cardiovascular damage following treatment with N(G)-nitro-L-arginine methyl ester plus angiotensin II an increased abundance of striatin protein in heart and kidney tissue. CONCLUSION: Our results provide evidence that increased striatin levels is a component of MR activation in the vasculature and suggest that regulation of striatin by ALDO may modulate estrogen's nongenomic effects.

mRNA and protein levels of FUS, EWSR1, and TAF15 are upregulated in liposarcoma.

Translocations or mutations of FUS, EWSR1, and TAF15 (FET) result in distinct genetic diseases. N-terminal translocations of any FET protein to a series of transcription factors yields chimeric proteins that contribute to sarcomagenesis, whereas mutations in the conserved COOH-terminal domain of wild-type FUS were recently shown to cause familial amyotrophic lateral sclerosis. We thus investigated whether the loss of one FUS allele by translocation in liposarcoma may be followed by mutations in either the remaining FUS allele or the paralogous EWSR1. Furthermore, we investigated the strength of the FET promoters and their contributions to sarcomagenesis given the proteins' frequent involvement in oncogenic translocations. We sequenced the respective genomic regions of both FUS and EWSR1 in 96 liposarcoma samples. Additionally, we determined FET transcript and protein levels in several liposarcoma cell lines. We did not observe sequence variations in either FUS or EWSR1. However, protein copy numbers reached an impressive 0.9 and 5.5 Mio of FUS and EWSR1 per tumor cell, respectively. Compared with adipose-derived stem cells, FUS and EWSR1 protein expression levels were elevated on average 28.6-fold and 7.3-fold, respectively. TAF15 mRNA levels were elevated on average 3.9-fold, although with a larger variation between samples. Interestingly, elevated TAF15 mRNA levels did not translate to strongly elevated protein levels, consistent with its infrequent occurrence as translocation partner in tumors. These results suggest that the powerful promoters of FET genes are predominantly responsible for the oncogenic effect of transcription factor translocations in sarcomas.

Development and characterization of HAT-sensitive Ewing tumour cells for immunotherapy.

BACKGROUND: Despite improvements in the treatment of patients with Ewing family tumours (EFT) during the past decades, the prognosis for patients with advanced disease is still unsatisfying. New treatment strategies have to be developed. MATERIALS AND METHODS: A hypoxanthine/aminopterin/thymidine (HAT)-sensitive EFT cell line was developed by repetitive treatment of the EFT cell line SK-N-MC with 8'-azaguanine (8AG). By using DNA microarrays, the gene expression profile of this cell line was characterized. Immunostimulatory activity was assessed by mixed lymphocyte/tumour cell culture (MLTC). Artificial fusion of tumour cells and dendritic cells was visualized by flow cytometry. RESULTS: After selection of 8AG-resistant cells, a cell line with high sensitivity for treatment with HAT was obtained. Expression of the X chromosome inactivation specific transcript XIST was higher in HAT-sensitive cells. Nevertheless, HAT-sensitive cells retained the EFT-associated gene expression profile. Moreover, in the presence of HAT, it was possible to use these cells without irradiation as stimulatory cells in MLTC or as fusion partner for dendritic cells. CONCLUSION: HAT-sensitive EFT cells might be an interesting tool for the development of new immunotherapeutic approaches for the treatment of EFT.

[Mesenchymal uterine tumors. Leiomyomas]. / Mesenchymale Uterustumoren. Leiomyogene Tumoren.

Leiomyomas are by far the most frequent mesenchymal uterine neoplasms. Leiomyoma variants refer to a particular histological differentiation and growth pattern, respectively. To assess malignancy, an algorithm is used based on the presence or absence of cellular atypia, tumor cell necrosis and mitosis. In addition, vascular invasion is a criterion for malignancy. Ischemic or infarct type necrosis is not considered a criterion for malignancy. The differential diagnosis of leiomyosarcoma includes cellular and mitotically active leiomyoma and leiomyoma with extensive infarct type necrosis (apoplectic leiomyoma). The term smooth muscle tumor of uncertain malignant potential (STUMP) should be reserved for tumors with uncertainty regarding cell type, type of necrosis and mitotic index, as well as for special cases of myxoid and epithelioid smooth muscle neoplasms. Immunohistochemically, the expression of desmin and caldesmon is indicative of smooth muscle tumors, while stromal tumors can express smooth muscle actin and, rarely, desmin in addition to CD10.

Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia.

Glucocorticoid receptor-mediated expression of caldesmon regulates cell migration via the reorganization of the actin cytoskeleton.

Glucocorticoids (GCs) play important roles in numerous cellular processes, including growth, development, homeostasis, inhibition of inflammation, and immunosuppression. Here we found that GC-treated human lung carcinoma A549 cells exhibited the enhanced formation of the thick stress fibers and focal adhesions, resulting in suppression of cell migration. In a screen for GC-responsive genes encoding actin-interacting proteins, we identified caldesmon (CaD), which is specifically up-regulated in response to GCs. CaD is a regulatory protein involved in actomyosin-based contraction and the stability of actin filaments. We further demonstrated that the up-regulation of CaD expression was controlled by glucocorticoid receptor (GR). An activated form of GR directly bound to the two glucocorticoid-response element-like sequences in the human CALD1 promoter and transactivated the CALD1 gene, thereby up-regulating the CaD protein. Forced expression of CaD, without GC treatment, also enhanced the formation of thick stress fibers and focal adhesions and suppressed cell migration. Conversely, depletion of CaD abrogated the GC-induced phenotypes. The results of this study suggest that the GR-dependent up-regulation of CaD plays a pivotal role in regulating cell migration via the reorganization of the actin cytoskeleton.