Myocardin-related transcription factor A (MRTF-A) activity-dependent cell adhesion is correlated to focal adhesion kinase (FAK) activity.

The regulation of cell-substrate adhesion is tightly linked to the malignant phenotype of tumor cells and plays a role in their migration, invasion, and metastasis. Focal adhesions (FAs) are dynamic adhesion structures that anchor the cell to the extracellular matrix. Myocardin-related transcription factors (MRTFs), co-regulators of the serum response factor (SRF), regulate expression of a set of genes encoding actin cytoskeletal/FA-related proteins. Here we demonstrated that the forced expression of a constitutively active MRTF-A (CA-MRTF-A) in B16F10 melanoma cells induced the up-regulation of actin cytoskeletal and FA proteins, resulting in FA reorganization and the suppression of cell migration. Expression of CA-MRTF-A markedly increased phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important components for FA dynamics. Notably, FAK activation was triggered by the clustering of up-regulated integrins. Our results revealed that the MRTF-SRF-dependent regulation of cell migration requires both the up-regulation of actin cytoskeletal/FA proteins and the integrin-mediated regulation of FA components via the FAK/Src pathway. We also demonstrated that activation of the MRTF-dependent transcription correlates FAK activation in various tumor cells. The elucidation of the correlation between MRTF and FAK activities would be an effective therapeutic target in focus of tumor cell migration.

Docosahexaenoic Acid Promotes Axon Outgrowth by Translational Regulation of Tau and Collapsin Response Mediator Protein 2 Expression.

n-3 PUFAs are essential for neuronal development and brain function. However, the molecular mechanisms underlying their biological effects remain unclear. Here we examined the mechanistic action of docosahexaenoic acid (DHA), the most abundant n-3 polyunsaturated fatty acids in the brain. We found that DHA treatment of cortical neurons resulted in enhanced axon outgrowth that was due to increased axon elongation rates. DHA-mediated axon outgrowth was accompanied by the translational up-regulation of Tau and collapsin response mediator protein 2 (CRMP2), two important axon-related proteins, and the activation of Akt and p70 S6 kinase. Consistent with these findings, rapamycin, a potent inhibitor of mammalian target of rapamycin (mTOR), prevented DHA-mediated axon outgrowth and up-regulation of Tau and CRMP2. In addition, DHA-dependent activation of the Akt-mTOR-S6K pathway enhanced 5'-terminal oligopyrimidine tract-dependent translation of Tau and CRMP2. Therefore, our results revealed an important role for the Akt-mTOR-S6K pathway in DHA-mediated neuronal development.

PSD-Zip70 Deficiency Causes Prefrontal Hypofunction Associated with Glutamatergic Synapse Maturation Defects by Dysregulation of Rap2 Activity.

Dysregulation of synapse formation and plasticity is closely related to the pathophysiology of psychiatric and neurodevelopmental disorders. The prefrontal cortex (PFC) is particularly important for executive functions such as working memory, cognition, and emotional control, which are impaired in the disorders. PSD-Zip70 (Lzts1/FEZ1) is a postsynaptic density (PSD) protein predominantly expressed in the frontal cortex, olfactory bulb, striatum, and hippocampus. Here we found that PSD-Zip70 knock-out (PSD-Zip70KO) mice exhibit working memory and cognitive defects, and enhanced anxiety-like behaviors. These abnormal behaviors are caused by impaired glutamatergic synapse transmission accompanied by tiny-headed immature dendritic spines in the PFC, due to aberrant Rap2 activation, which has roles in synapse formation and plasticity. PSD-Zip70 modulates the Rap2 activity by interacting with SPAR (spine-associated RapGAP) and PDZ-GEF1 (RapGEF) in the postsynapse. Furthermore, suppression of the aberrant Rap2 activation in the PFC rescued the behavioral defects in PSD-Zip70KO mice. Our data demonstrate a critical role for PSD-Zip70 in Rap2-dependent spine synapse development in the PFC and underscore the importance of this regulation in PFC-dependent behaviors. SIGNIFICANCE STATEMENT: PSD-Zip70 deficiency causes behavioral defects in working memory and cognition, and enhanced anxiety due to prefrontal hypofunction. This study revealed that PSD-Zip70 plays essential roles in glutamatergic synapse maturation via modulation of the Rap2 activity in the PFC. PSD-Zip70 interacts with both SPAR (spine-associated RapGAP) and PDZ-GEF1 (RapGEF) and modulates the Rap2 activity in postsynaptic sites. Our results provide a novel Rap2-specific regulatory mechanism in synaptic maturation involving PSD-Zip70.

Diversification of caldesmon-linked actin cytoskeleton in cell motility.

The actin cytoskeleton plays a key role in regulating cell motility. Caldesmon (CaD) is an actin-linked regulatory protein found in smooth muscle and non-muscle cells that is conserved among a variety of vertebrates. It binds and stabilizes actin filaments, as well as regulating actin-myosin interaction in a calcium (Ca2+)/calmodulin (CaM)- and/or phosphorylation-dependent manner. CaD function is regulated qualitatively by Ca2+/CaM and by its phosphorylation state and quantitatively at the mRNA level, by three different transcriptional regulation of the CALD1 gene. CaD has numerous functions in cell motility, such as migration, invasion, and proliferation, exerted via the reorganization of the actin cytoskeleton. Here we will outline recent findings regarding CaD's structural features and functions.

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.

Dual roles of myocardin-related transcription factors in epithelial mesenchymal transition via slug induction and actin remodeling.

Epithelial-mesenchymal transition (EMT) is a critical process occurring during embryonic development and in fibrosis and tumor progression. Dissociation of cell-cell contacts and remodeling of the actin cytoskeleton are major events of the EMT. Here, we show that myocardin-related transcription factors (MRTFs; also known as MAL and MKL) are critical mediators of transforming growth factor beta (TGF-beta) 1-induced EMT. In all epithelial cell lines examined here, TGF-beta1 triggers the nuclear translocation of MRTFs. Ectopic expression of constitutive-active MRTF-A induces EMT, whereas dominant-negative MRTF-A or knockdown of MRTF-A and -B prevents the TGF-beta1-induced EMT. MRTFs form complexes with Smad3. Via Smad3, the MRTF-Smad3 complexes bind to a newly identified cis-element GCCG-like motif in the promoter region of Canis familiaris and the human slug gene, which activates slug transcription and thereby dissociation of cell-cell contacts. MRTFs also increase the expression levels of actin cytoskeletal proteins via serum response factor, thereby triggering reorganization of the actin cytoskeleton. Thus, MRTFs are important mediators of TGF-beta1-induced EMT.

Changes in the balance between caldesmon regulated by p21-activated kinases and the Arp2/3 complex govern podosome formation.

Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.