Cyclin D1 Functions in Cell Migration

Cell migration is essential for developmental morphogenesis, tissue repair, and tumor metastasis. A recent study reveals that cyclin D1 acts to promote cell migration by inhibiting Rho/ROCK signaling and expression of thrombospondin-1 (TSP-1), an extracellular matrix protein that regulates cell migration in many settings including cancer. Given the frequent overexpression of cyclin D1 in cancer cells, due to its upregulation by Ras, Rho, Src, and other genes that drive malignant development, the new findings suggest that cyclin D1 may have a central role in mediating invasion and metastasis of cancer cells by controlling Rho/ROCK signaling and matrix deposition of TSP-1.     

Cyclin D1 governs adhesion and motility of macrophages

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein, thereby promoting cell-cycle progression. Cyclin D1 is overexpressed in hematopoetic and epithelial malignancies correlating with poor prognosis and metastasis in several cancer types. Because tumor-associated macrophages have been shown to enhance malignant progression and metastasis, and cyclin D1-deficient mice are resistant to oncogene-induced malignancies, we investigated the function of cyclin D1-/- bone marrow-derived macrophages. Cyclin D1 deficiency increased focal complex formation at the site of substratum contact, and enhanced macrophage adhesion, yielding a flattened, circular morphology with reduced membrane ruffles. Migration in response to wounding, cytokine-mediated chemotaxis, and transendothelial cell migration of cyclin D1-/- bone marrow-derived macrophages were all substantially reduced. Thus, apart from proliferative and possible motility defects in the tumor cells themselves, the reduced motility and invasiveness of cyclin D1-/- tumor-associated macrophages may contribute to the tumor resistance of these mice.     

PACSIN 2 represses cellular migration through direct association with cyclin D1 but not its alternate splice form cyclin D1b

Cyclin D1 overexpression is a common feature of many human malignancies. Genomic deletion analysis has demonstrated a key role for cyclin D1 in cellular proliferation, angiogenesis and cellular migration. To investigate the mechanisms contributing to cyclin D1 functions, we purified cyclin D1a-associated complexes by affinity chromatography and identified the PACSIN 2 (protein kinase C and casein kinase substrate in neurons 2) protein by mass spectrometry. The PACSIN 2, but not the related PACSIN 1 and 3, directly bound wild-type cyclin D1 (cyclin D1a) at the carboxyl terminus and failed to bind cyclin D1b, the alternative splicing variant of cyclin D1. PACSIN 2 knockdown induced cellular migration and reduced cell spreading in LNCaP cells expressing cyclin D1a. In cyclin D1^−/− mouse embryonic fibroblasts (MEFs), cyclin D1a, but not cyclin D1b, reduced the cell spreading to a polarized morphology. siPACSIN 2 had no effect on cellular migration of cyclin D1^−/− MEFs. Cyclin D1a restored the migratory ability of cyclin D1^−/− MEFs, which was further enhanced by knocking down PACSIN 2 with siRNA. The cyclin D1-associated protein, PACSIN 2, regulates cell spreading and migration, which are dependent on cyclin D1 expression.Key words: PACSIN 2, cyclin D1, polymorphism, cellular migration, cell spreading, cancer     

Cyclin D1 inhibits peroxisome proliferator-activated receptor gamma-mediated adipogenesis through histone deacetylase recruitment

The cyclin D1 gene encodes the labile serum-inducible regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. Overexpression of cyclin D1 promotes cellular proliferation and normal physiological levels of cyclin D1 function to inhibit adipocyte differentiation in vivo. We have previously shown that cyclin D1 inhibits peroxisome proliferator-activated receptor (PPAR)gamma-dependent activity through a cyclin-dependent kinase- and retinoblastoma protein-binding-independent mechanism. In this study, we determined the molecular mechanism by which cyclin D1 regulated PPARgamma function. Herein, murine embryonic fibroblast (MEF) differentiation by PPARgamma ligand was associated with a reduction in histone deacetylase (HDAC1) activity. Cyclin D1-/- MEFs showed an increased propensity to undergo differentiation into adipocytes. Genetic deletion of cyclin D1 reduced HDAC1 activity. Reconstitution of cyclin D1 into the cyclin D1-/- MEFs increased HDAC1 activity and blocked PPARgamma-mediated adipogenesis. PPARgamma activity was enhanced in cyclin D1-/- cells. Reintroduction of cyclin D1 inhibited basal and ligand-induced PPARgamma activity and enhanced HDAC repression of PPARgamma activity. Cyclin D1 bound HDAC in vivo and preferentially physically associated with HDAC1, HDAC2, HDAC3, and HDAC5. Chromatin immunoprecipitation assay demonstrated that cyclin D1 enhanced recruitment of HDAC1 and HDAC3 and histone methyltransferase SUV39H1 to the PPAR response element of the lipoprotein lipase promoter and decreased acetylation of total histone H3 and histone H3 lysine 9. Collectively, these studies suggest an important role of cyclin D1 in regulation of PPARgamma-mediated adipocyte differentiation through recruitment of HDACs to regulate PPAR response element local chromatin structure and PPARgamma function.     

Regulation of RhoA-dependent ROCKII activation by Shp2

Contractile forces mediated by RhoA and Rho kinase (ROCK) are required for a variety of cellular processes, including cell adhesion. In this study, we show that RhoA-dependent ROCKII activation is negatively regulated by phosphorylation at a conserved tyrosine residue (Y722) in the coiled-coil domain of ROCKII. Tyrosine phosphorylation of ROCKII is increased with cell adhesion, and loss of Y722 phosphorylation delays adhesion and spreading on fibronectin, suggesting that this modification is critical for restricting ROCKII-mediated contractility during these processes. Further, we provide evidence that Shp2 mediates dephosphorylation of ROCKII and, therefore, regulates RhoA-induced cell rounding, indicating that Shp2 couples with RhoA signaling to control ROCKII activation during deadhesion. Thus, reversible tyrosine phosphorylation confers an additional layer of control to fine-tune RhoA-dependent activation of ROCKII.     

Genetic reevaluation of the role of F-box proteins in cyclin D1 degradation

D-type cyclins play a pivotal role in G(1)-S progression of the cell cycle, and their expression is frequently deregulated in cancer. Cyclin D1 has a half-life of only ~30 min as a result of its ubiquitylation and proteasomal degradation, with various F-box proteins, including Fbxo4, Fbxw8, Skp2, and Fbxo31, having been found to contribute to its ubiquitylation. We have now generated Fbxo4-deficient mice and found no abnormalities in these animals. Cyclin D1 accumulation was thus not observed in Fbxo4(-/-) mouse tissues. The half-life of cyclin D1 in mouse embryonic fibroblasts (MEFs) prepared from Fbxo4(-/-), Fbxw8(-/-), and Fbxo4(-/-); Fbxw8(-/-) mice also did not differ from that in wild-type MEFs. Additional depletion of Skp2 and Fbxo31 in Fbxo4(-/-); Fbxw8(-/-) MEFs by RNA interference did not affect cyclin D1 stability. Although Fbxo31 depletion in MEFs increased cyclin D1 abundance, this effect appeared attributable to upregulation of cyclin D1 mRNA. Furthermore, abrogation of the function of the Skp1-Cul1-F-box protein (SCF) complex or the anaphase-promoting complex/cyclosome (APC/C) complexes did not alter the half-life of cyclin D1, whereas cyclin D1 degradation was dependent largely on proteasome activity. Our genetic analyses thus do not support a role for any of the four F-box proteins examined in cyclin D1 degradation during normal cell cycle progression. They suggest the existence of other ubiquitin ligases that target cyclin D1 for proteolysis.     

ROCKII Ser1366 phosphorylation reflects the activation status

ROCK (Rho-associated protein kinase), a downstream effector of RhoA, plays an important role in many cellular processes. Accumulating evidence has shown the involvement of ROCK activation in the pathogenesis of many diseases. However, a reagent capable of detecting ROCK activation directly is lacking. In the present study, we show autophosphorylation of ROCKII in an in vitro kinase reaction. The phosphorylation sites were identified by MS, and the major phosphorylation site was found to be at the highly conserved residue Ser1366. A phospho-specific antibody was generated that can specifically recognize ROCKII Ser1366 phosphorylation. We found that the extent of Ser1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that Ser1366 phosphorylation reflects its kinase activity. In addition, ROCKII Ser1366 phosphorylation could be detected in human breast tumours by immunohistochemical staining. The present study provides a new approach for revealing the ROCKII activation status by probing ROCKII Ser1366 phosphorylation directly in cells or tissues.     

Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

Endometriosis is a disease characterized by the localization of endometrial tissue outside the uterine cavity. The differences observed in migration of human endometrial stromal cells (hESC) obtained from patients with endometriosis versus healthy controls were proposed to correlate with the abnormal activation of Raf-1/ROCKII signalling pathway. To evaluate the mechanism by which Raf-1 regulates cytoskeleton reorganization and motility, we used primary eutopic (Eu-, n = 16) and ectopic (Ec-, n = 8; isolated from ovarian cysts) hESC of patients with endometriosis and endometriosis-free controls (Co-hESC, n = 14). Raf-1 siRNA knockdown in Co- and Eu-hESC resulted in contraction and decreased migration versus siRNA controls. This phenotype was reversed following the re-expression of Raf-1 in these cells. Lowest Raf-1 levels in Ec-hESC were associated with hyperactivated ROCKII and ezrin/radixin/moesin (E/R/M), impaired migration and a contracted phenotype similar to Raf-1 knockdown in Co- and Eu-hESC. We further show that the mechanism by which Raf-1 mediates migration in hESC includes direct myosin light chain phosphatase (MYPT1) phosphorylation and regulation of the levels of E/R/M, paxillin, MYPT1 and myosin light chain (MLC) phosphorylation indirectly via the hyperactivation of ROCKII kinase. Furthermore, we suggest that in contrast to Co-and Eu-hESC, where the cellular Raf-1 levels regulate the rate of migration, the low cellular Raf-1 content in Ec-hESC, might ensure their restricted migration by preserving the contracted cellular phenotype. In conclusion, our findings suggest that cellular levels of Raf-1 adjust the threshold of hESC migration in endometriosis.     

Differential regulation of adhesion complex turnover by ROCK1 and ROCK2

Background

ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration.

Methodology/Principal Findings

In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration.

Conclusions

ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes.     

B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts

Recent data have shown that the BRAF gene is mutated at a high frequency in human malignancies. We have analyzed the migratory characteristics of B-raf(-/-) mouse embryonic fibroblasts (MEFs) and compared these with the organization of the actin cytoskeleton and the activity of signaling pathways that are known to influence this organization. Disruption of B-raf significantly reduced the levels of phospho-ERK1/2 and, surprisingly, induced an approximately 1.5-fold increase in cell migration. Consistent with these findings, the high level of actin stress fibers normally present in MEFs was considerably reduced following disruption of B-raf, and the F-actin content of B-raf(-/-) cells was less than half that of B-raf(+/+) cells. Phosphorylation of the myosin light chain on Thr18/Ser19 residues was not reduced in B-raf(-/-) cells. Rather, reduced ROCKII expression and attenuated phosphorylation of ADF/cofilin on serine 3 occurred. Normal stress fiber and phosphocofilin levels were restored by the expression of human B-Raf and catalytically active MEK and by the overexpression of LIM kinase (LIMK). These results have important implications for the role of the B-Raf/ERK signaling pathway in regulating cell motility in normal and malignant cells. They suggest that B-Raf is involved in invasiveness by regulating the proper assembly of actin stress fibers and contractility through a ROCKII/LIMK/cofilin signaling pathway.     

Regulation of the Src Kinase-associated Phosphoprotein 55 Homologue by the Protein Tyrosine Phosphatase PTP-PEST in the Control of Cell Motility

PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.     

Lysophosphatidic acid regulates the motility of MCF10CA1a breast cancer cell sheets via two opposing signaling pathways

Aberrant cell migration leads to the dispersal of malignant cells. The ubiquitous lipid mediator lysophosphatidic acid (LPA) modulates cell migration and is implicated in tumor progression. Yet, the signaling cascades that regulate LPA's effect on cell motility remain unclear. Using time-lapse imaging and quantitative analyses, we studied the role of signaling cascades that act downstream of LPA on the motility of MCF10CA1a breast cancer cells. We found that LPA alters cell motility via two major signaling pathways. The Rho/ROCK signaling cascade is the predominant pathway that increases E-Cadherin containing cell–cell adhesions and cortical arrangement of actomyosin to promote slow, directional, spatially coherent and temporally consistent movement. In contrast, Gα_i/o- and Gα_q/11-dependent signaling cascades lessen directionality and support the independent movement of cells. The net effect of LPA on breast cancer cell migration therefore results from the integrated signaling activity of the Rho/ROCK and Gα_i/o- and Gα_q/11-dependent pathways, thus allowing for a dynamic migratory response to changes in the cellular or microenvironmental context.     

Deleted in liver cancer 1 (DLC1) negatively regulates Rho/ROCK/MLC pathway in hepatocellular carcinoma

Aims

Deleted in liver cancer 1 (DLC1), a member of RhoGTPase activating protein (GAP) family, is known to have suppressive activities in tumorigenicity and cancer metastasis. However, the underlying molecular mechanisms of how DLC1 suppresses cell motility have not been fully elucidated. Rho-kinase (ROCK) is an immediate down-stream effector of RhoA in mediating cellular cytoskeletal events and cell motility. In the present study, we aimed to investigate the effects of DLC1 on Rho/ROCK signaling pathway in hepatocellular carcinoma (HCC).

Methodology/Principal Findings

We demonstrated that DLC1 negatively regulated ROCK-dependent actomyosin contractility. From immumofluorescence study, we found that ectopic expression of DLC1 abrogated Rho/ROCK-mediated cytoskeletal reorganization including formation of stress fibers and focal adhesions. It also downregulated cortical phosphorylation of myosin light chain 2 (MLC2). These inhibitory events by DLC1 were RhoGAP-dependent, as RhoGAP-deficient mutant of DLC1 (DLC1 K714E) abolished these inhibitory events. In addition, from western study, DLC1 inhibited ROCK-related myosin light chain phosphatase targeting unit 1 (MYPT1) phosphorylation at Threonine 853. By examining cell morphology under microscope, we found that ectopic expression of dominant-active ROCK released cells from DLC1-induced cytoskeletal collapse and cell shrinkage.

Conclusion

Our data suggest that DLC1 negatively regulates Rho/ROCK/MLC2. This implicates a ROCK-mediated pathway of DLC1 in suppressing metastasis of HCC cells and enriches our understanding in the molecular mechanisms involved in the progression of hepatocellular carcinoma.     

Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility

Rho GTPases participate in various cellular processes, including normal and tumor cell migration. It has been reported that RhoA is targeted for degradation at the leading edge of migrating cells by the E3 ubiquitin ligase Smurf1, and that this is required for the formation of protrusions. We report that Smurf1-dependent RhoA degradation in tumor cells results in the down-regulation of Rho kinase (ROCK) activity and myosin light chain 2 (MLC2) phosphorylation at the cell periphery. The localized inhibition of contractile forces is necessary for the formation of lamellipodia and for tumor cell motility in 2D tissue culture assays. In 3D invasion assays, and in in vivo tumor cell migration, the inhibition of Smurf1 induces a mesenchymal-amoeboid-like transition that is associated with a more invasive phenotype. Our results suggest that Smurf1 is a pivotal regulator of tumor cell movement through its regulation of RhoA signaling.     

Rho GTPase-dependent signaling is required for macrophage migration inhibitory factor-mediated expression of cyclin D1

Our previous studies demonstrated that the proinflammatory peptide, macrophage migration inhibitory factor (MIF), functions as an autocrine mediator of both growth factor- and integrin-dependent sustained ERK MAPK activation, cyclin D1 expression, and cell cycle progression. We now report that MIF promotes the activation of the canonical ERK MAPK cascade and cyclin D1 expression by stimulating the activity of the Rho GTPase and downstream signaling to stress fiber formation. Rho-dependent stress fiber accumulation promotes the sustained activation of ERK and subsequent cyclin D1 expression during G(1)-S phase cell cycle progression. This pathway is reported to be dependent upon myosin light chain (MLC) kinase, integrin clustering, and subsequent activation of focal adhesion kinase, leading to sustained MAPK activity. Our studies reveal that recombinant MIF induces cyclin D1 expression in a Rho-, Rho kinase-, MLC kinase-, and ERK-dependent manner in asynchronous NIH 3T3 fibroblasts. Moreover, MIF(-/-) murine embryonic fibroblasts display aberrant cyclin D1 expression that is linked to defective Rho activity, stress fiber formation, and MLC phosphorylation. These results suggest that MIF is an integral autocrine mediator of Rho GTPase-dependent signaling events and provide mechanistic insight into how MIF regulates proliferative, migratory, and oncogenic processes.     

Rho-associated kinase (ROCK) inhibition reverses low cell activity on hydrophobic surfaces

Hydrophobic polymers do not offer an adequate scaffold surface for cells to attach, migrate, proliferate, and differentiate. Thus, hydrophobic scaffolds for tissue engineering have traditionally been physicochemically modified to enhance cellular activity. However, modifying the surface by chemical or physical treatment requires supplementary engineering procedures. In the present study, regulation of a cell signal transduction pathway reversed the low cellular activity on a hydrophobic surface without surface modification. Inhibition of Rho-associated kinase (ROCK) by Y-27632 markedly enhanced adhesion, migration, and proliferation of osteoblastic cells cultured on a hydrophobic polystyrene surface. ROCK inhibition regulated cell-cycle-related molecules on the hydrophobic surface. This inhibition also decreased expression of the inhibitors of cyclin-dependent kinases such as p21^cip1 and p27^kip1 and increased expression of cyclin A and D. These results indicate that defective cellular activity on the hydrophobic surface can be reversed by the control of a cell signal transduction pathway without physicochemical surface modification.     

Glycine-extended gastrin stimulates cell proliferation and migration through a Rho- and ROCK-dependent pathway, not a Rac/Cdc42-dependent pathway

Both amidated gastrin (Gamide) and glycine-extended gastrin (Ggly) stimulate gastrointestinal cell proliferation and migration. Binding of Gamide to the cholecystokinin-2 receptor activates small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42), and dominant-negative mutants of Rho or Cdc42 block Gamide-stimulated cell proliferation and survival. In comparison, little is known about the Ggly signaling transduction pathway leading to cell proliferation and migration. The present study examined the roles of the small G proteins Rho, Rac, and Cdc42 in Ggly-induced proliferation and migration of the mouse gastric epithelial cell line IMGE-5. Ggly stimulated the activation of Rho and its downstream effector protein ROCK. The activation of Rho and ROCK mediated Ggly-induced cell proliferation and migration as inhibition of Rho by C3, or ROCK by Y-27632, completely blocked these effects of Ggly. Ggly also stimulated tyrosine phosphorylation of focal adhesion kinase, and stimulation was reversed by addition of C3 and Y-27632. In contrast to the effects of Rho and ROCK, inhibition of the Rac or Cdc42 pathways by expression of dominant-negative mutants of Rac or Cdc42 did not affect Ggly-induced cell proliferation and migration. These results demonstrate that Ggly stimulates IMGE-5 cell proliferation and migration through a Rho/ROCK-dependent pathway but not via Rac- or Cdc42-dependent pathways.