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Actin is a highly abundant cytoskeletal protein that is essential for all eukaryotic cells and participates in many structural and functional roles. It has long been noted that estrogen affects cellular morphology. However, recent studies observed that both estrogen and tamoxifen induce a remarkable cytoskeletal remodeling independent of ER. In addition to ER, G protein-coupled estrogen receptor 1 (GPER, also known as GPR30) also binds to estrogen with high affinity and mediates intracellular estrogenic signaling. Here, we show that activation of GPER by its specific agonist G-1 induces re-organization of F-actin cytoskeleton. We further demonstrate that GPER acts through PLCβ-PKC and Rho/ROCK-LIMK-Cofilin pathway, which are upstream regulators of F-actin cytoskeleton assembly, thereby enhancing TAZ nuclear localization and activation. Furthermore, we find that LIMK1/2 is critical for GPER activation-induced breast cancer cell migration. Together, our results suggest that GPER mediates G-1-induced cytoskeleton assembly and GPER promotes breast cancer cell migration via PLCβ-PKC and Rho/ROCK-LIMK-Cofilin pathway.

Intracranial aneurysm, as a common cause of cerebral hemorrhage, is often discovered when the aneurysm ruptures, causing subarachnoid hemorrhage. Unfortunately, the formation of cerebral aneurysm, which is associated with endothelial damage and macrophage migration, still cannot be prevented now. Tight junctions (TJs) open due to the disappearance of TJ proteins occludin and zona occludens-1 (ZO-1) in damaged endothelia, thus allowing macrophage migration and forming cerebral aneurysm. Therefore, cerebral aneurysm formation can be prevented by increasing TJs of the artery endothelium. Interestingly, statin, which can reduce saccular aneurysm, may prevent aneurysm formation through acting on different steps, but the underlying mechanism remains unclear. In this study, angiotensin II (Ang II) significantly increased the permeability of human arterial endothelial cell (HAEC). Moreover, the distribution of ZO-1 in cell-cell junction area and the total expression in HAECs were significantly decreased by Ang II treatment. However, the abnormal distribution and decreased expression of ZO-1 and hyperpermeability of HAECs were significantly reversed by pretreatment with atorvastatin. Furthermore, Ang II-induced phosphorylations of MYPT1, LIMK and MLC2 were significantly inhibited with atorvastatin or Rho kinase (ROCK) inhibitor (H1152) pretreatment. Knockdown of ROCK-II probably abolished Ang II-induced abnormal ZO-1 distribution and expression deficiency and hyperpermeability of HAECs. In conclusion, atorvastatin prevented Ang II-induced rupture of HAEC monolayers by suppressing the ROCK signaling pathway. Our results may explain, at least in part, some beneficial effects of statins on cardiovascular diseases such as intracranial aneurysm.

Estrogen is a key factor to induce the sexually dimorphic nucleus (SDN) in the preoptic area (POA) of the rat brain. Identification of estrogen-dependent signaling pathways at SDN in POA during the critical period is a prerequisite for elucidating the mechanism. In the present study, we treated female rats with/without 17β-estradiol (E2) at birth, designated as postnatal day 1 (P1), and prepared total RNA from brain slices containing SDN for DNA microarray analysis. Among the estrogen-responsive genes identified, protein kinase C-delta (PKC-δ) was significantly up-regulated by E2 at P5. We examined the downstream effectors of PKC-δ protein by Western blotting and found an E2-induced PKC-δ/Rac1/PAK1/LIMK1/cofilin pathway. In the pathway, E2 suppressed the phosphorylation (inactive form) of cofilin. This result was supported by immunohistochemistry, where the phosphorylation/dephosphorylation of cofilin occurred at SDN, which suggests that cell migration is a cue to create sexual dimorphism in POA.

Resveratrol has been reported to suppress cancer progression in several in vivo and in vitro models, whereas ultraviolet B (UVB), a major risk for skin cancer, is known to induce cell death in cancerous cells. Here, we investigated whether resveratrol can sensitize A431 human epidermoid carcinoma cells to UVB-induced cell death. We examined the combined effect of UVB (30 mJ/cm(2)) and resveratrol (60 microM) on A431 cells. Exposure of A431 carcinoma cells to UVB radiation or resveratrol can inhibit cell proliferation and induce apoptosis. However, the combination of resveratrol and UVB exposure was associated with increased proliferation inhibition of A431 cells compared with either agent alone. Furthermore, results showed that resveratrol and UVB treatment of A431 cells disrupted the nuclear factor-kappaB (NF-kappaB) pathway by blocking phosphorylation of serine 536 and inactivating NF-kappaB and subsequent degradation of IkappaBalpha, which regulates the expression of survivin. Resveratrol and UVB treatment also decreased the phosphorylation of tyrosine 701 of the important transcription factor signal transducer activator of transcription (STAT1), which in turn inhibited translocation of phospho-STAT1 to the nucleus. Moreover, resveratrol/UVB also inhibited the metastatic protein LIMK1, which reduced the motility of A431 cells. In conclusion, our study demonstrates that the combination of resveratrol and UVB act synergistically against skin cancer cells. Thus, resveratrol is a potential chemotherapeutic agent against skin carcinogenesis.

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor-beta superfamily. BMPs regulate several crucial aspects of embryonic development and organogenesis. Here, we demonstrate that BMP-2 inhibits the neurite outgrowth of postnatal cerebellar neurons in vitro. Although receptor-regulated Smad proteins are activated by BMP-2, this signal transduction is not necessary for the inhibitory effect of BMP-2. Interestingly, BMP-2 activates LIM-kinase 1 in the neurons, and the dominant negative form of LIM-kinase 1 abolishes the effect of BMP-2. Thus, BMP-2 inhibits neurite outgrowth by a LIM-kinase 1-dependent mechanism, and our findings add a new member to the group of neurite growth inhibitors.

Stimulation of rat renal mesangial cells with cell-permeable C6-ceramide for 6 and 24 h induces the expression of several genes as analyzed by a RNA fingerprinting arbitrarily primed-PCR method. Sequencing of the differentially expressed bands identified the serine/threonine protein kinase LIM kinase-1 (LIMK-1), which is involved in the regulation of cytoskeletal organization, as a ceramide-induced gene. The ceramide-triggered upregulation of LIMK-1 was verified by semiquantitative reverse transcriptase-PCR. A detailed time course reveals a first detectable increase in RNA level after 2 h of ceramide stimulation which reaches maximal levels after 6 h of stimulation and remains elevated up to 24 h. This ceramide-induced gene transcription of LIMK-1 is accompanied by enhanced LIMK-1 protein levels with maximal protein expression seen after 6 h of stimulation. Furthermore, cofilin, which is a specific substrate of LIMK-1, shows an increased phosphorylation at Ser-3 in mesangial cells exposed to C6-ceramide. Mechanistically, the ceramide-induced LIMK-1 expression is blocked by the Rho kinase inhibitor Y27632, but not by a farnesyl transferase inhibitor, suggesting the involvement of the small G protein Rho, but not Ras and Rac, in the expressional upregulation. Similar to exogenously added ceramide, also interleukin-1β which is an established activator of the neutral sphingomyelinase that leads to endogenous ceramide formation upregulates LIMK-1 protein expression and activity. In summary, these data demonstrate for the first time that LIMK-1 is a ceramide-induced gene, thus suggesting that LIMK-1 may act as a link between stress-induced ceramide formation and reorganization of the actin cytoskeleton.

LIM-kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylation of cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cell-cycle-dependent activity of endogenous LIMK1. When HeLa cells were synchronized at prometaphase by nocodazole-treatment, LIMK1 was hyperphosphorylated, and its activity toward cofilin phosphorylation was markedly increased. During cell cycle progression, LIMK1 activity was low in interphase but reached a maximal level during mitosis. Activation of LIMK1 during mitosis was abrogated by roscovitine, a specific inhibitor of cyclin-dependent kinases (CDKs), suggesting that activation of CDKs directly or indirectly participates in LIMK1 activation. These results strongly suggest that LIMK1 may play an important role in the cell cycle progression through regulation of actin cytoskeletal rearrangements.

Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous-transformed human RPE cells undergo cytoskeletal rearrangements via Rac1 GTPase-dependent pathways that modulate LIMK1 and cofilin activity. The TGFβ-like activity of the vitreous may participate in this effect. Actin polymerization causes the cytoskeletal rearrangements that lead to the plasticity of vitreous-transformed RPE cells in PVR.

Tumor cells can migrate in 3D matrices in either a mesenchymal-like or amoeboid mode. HT1080 fibrosarcoma cells cultured in 3D collagen gels change their morphology from mesenchymal-like (elongated) to amoeboid (round) following protease inhibitor (PI) treatment or active Rho or ROCK expression. In this study, we examined the role of LIM-kinase 1 (LIMK1) in the PI- or Rho/ROCK-induced cell morphological change. We showed that LIMK1 was activated after PI treatment of HT1080 cells in 3D collagen gels and this activation was blocked by a ROCK inhibitor. While overexpression of LIMK1 induced cell rounding, knockdown of LIMK1 or the expression of kinase-inactive LIMK1 suppressed PI- or Rho/ROCK-induced cell rounding. These results suggest that LIMK1 plays an essential role in the PI- or Rho/ROCK-induced mesenchymal-to-amoeboid cell morphological transition of HT1080 cells cultured in 3D collagen gels. Furthermore, LIMK1 knockdown suppressed the invasive activity of HT1080 cells in collagen gels with or without PIs, indicating that LIMK1 mediates both the mesenchymal and amoeboid modes of invasion of HT1080 cells.

Mammalian LIM-kinases (LIMKs) phosphorylate cofilin and induce actin cytoskeletal reorganization. To elucidate the functional roles of LIMKs in vivo during developmental processes, we attempted to isolate the cDNA encoding a Drosophila homolog of LIMK (DLIMK) and identified two isoforms of DLIMK transcripts coding for proteins with 1235 and 1257 amino acids, possessing the structure composed of two LIM domains, a PDZ domain, a protein kinase domain, and an unusual long C-terminal extension. In situ hybridization analysis in Drosophila embryos detected the uniformly distributed DLIMK mRNA in stages 2 to 5. In vitro kinase reaction revealed that DLIMK efficiently phosphorylates Drosophila cofilin (twinstar) specifically at Ser-3, the site responsible for inactivation of its actin-depolymerizing activity. When expressed in cultured cells, wild-type DLIMK, but not its kinase-inactive form, induced changes in actin cytoskeletal organization. These observations suggest that the LIMK-cofilin signaling pathway for regulating actin filament dynamics is evolutionarily conserved between Drosophila and mammals.

LIM kinases, composed of LIMK1 and LIMK2, have unique structural features that contain two LIM motifs at the N-terminus and a catalytic domain at the C-terminus. We report evidence of a novel type of mouse LIMK2 (Limk2) transcript specifically expressed in testis. cDNA cloning showed this Limk2 variant, designated tLimk2, lacked LIM domains at the N-terminus, due to usage of a testis-specific, alternative initiation exon. In Northern blot analysis, tLimk2 was detected in intact adult testis, but not in germ-cell-deficient or immature testis, indicating the stage-specific expression of tLimk2 in spermatogenic cells. In situ hybridization clearly demonstrated that tLimk2 was restrictedly expressed in differentiated germ cells (pachytene spermatocytes to round spermatids) and not expressed in early stages of spermatogenic cells and somatic cells in testis. These results suggested the possibility that the tLimk2 product is involved in spermatogenesis, especially in meiotic and/or postmeiotic processes.

LIM-kinase 1 (LIMK1) and LIM-kinase 2 (LIMK2) are members of a novel serine/threonine kinase subfamily with structural features composed of N-terminal two LIM domains, an internal PDZ-like domain, and a C-terminal protein kinase domain. We recently identified and characterized the mouse Limk2 gene and two Limk2 transcripts (Limk2a and Limk2b) coding for proteins with distinct N-terminal LIM structures. Here we describe two additional transcripts of the mouse Limk2 gene. One is a 1.7-kb transcript, termed Limk2t, which is specifically expressed in the testis and codes for an N-terminally truncated form of LIMK2 consisting of only a part of a PDZ-like domain and a protein kinase domain. The other is a transcript, termed Limk2c, which is specifically expressed in the brain and codes for a protein with a 6-amino-acid insert within the protein kinase domain. Exons specific to the 5'-terminal extra sequence of Limk2t and the insert sequence of Limk2c locate between exons 5-6 and exons 8-9 in the mouse Limk2 gene, respectively. Testis- and brain-specific expression of Limk2t and Limk2c suggests specific roles in these tissues.

In our efforts to explore possible roles of proteins with a LIM domain, which is a cysteine-rich Zinc-binding motif, in differentiation and oncogenesis in the lung, we have cloned a human LIMK2 gene and identified two alternative transcripts, LIMK2a and LIMK2b, which are probably due to variation in transcriptional initiation. The former encodes a protein containing two LIM domains, a PDZ domain, and a kinase domain, while the latter has only one and half LIM domains. The predominance of the two transcripts appears to be regulated in a tissue-specific manner. Alteration of the regulation is also observed in some cancer cell lines. Transfection studies have shown an association of 63-kDa and 58-kDa proteins with the LIMK2a and LIMK2b protein; the former is distributed in the cytoplasm and nucleus and the latter occurs mainly in the cytoplasm and is scarcely translocated to the nucleus. In contrast, a truncated LIMK2-Kinase has a nuclear location, not showing the protein association.