Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages.

Podosomes, adhesion structures capable of matrix degradation, have been linked with the ability of cells to perform chemotaxis and invade tissues. Wiskott-Aldrich Syndrome protein (WASp), an effector of the RhoGTPase Cdc42 and a Src family kinase substrate, regulates macrophage podosome formation. In this study, we demonstrate that WASp is active in podosomes by using TIRF-FRET microscopy. Pharmacological and RNA interference approaches suggested that continuous WASp activity is required for podosome formation and function. Rescue experiments using point mutations demonstrate an absolute requirement for Cdc42 binding to WASp in podosome formation. Although tyrosine phosphorylation was not absolutely required for podosome formation, phosphorylation did regulate the rate of podosome nucleation and actin filament stability. Importantly, WASp tyrosine phosphorylation does not alter WASp activation, instead phosphorylation appears to be important for the restriction of WASp activity to podosomes. In addition, the matrix-degrading ability of cells requires WASp phosphorylation. Chemotactic responses to CSF-1 were also attenuated in the absence of endogenous WASp, which could not be rescued with either tyrosine mutation. These results suggest a more complex role for tyrosine phosphorylation than simply in the regulation of WASp activity, and suggest a link between podosome dynamics and macrophage migration.

N-WASP has the ability to compensate for the loss of WASP in macrophage podosome formation and chemotaxis.

Wiskott-Aldrich syndrome protein (WASP) and its homologue neural-WASP (N-WASP) are nucleation promoting factors that integrate receptor signaling with actin cytoskeleton rearrangement. While hematopoietic cells express both WASP and N-WASP, WASP deficiency results in altered cell morphology, loss of podosomes and defective chemotaxis. It was determined that cells from a mouse derived monocyte/macrophage cell line and primary cells of myeloid lineage expressed approximately 15-fold higher levels of WASP relative to N-WASP. To test whether N-WASP can compensate for the loss of WASP and restore actin cytoskeleton integrity, N-WASP was overexpressed in macrophages, in which endogenous WASP expression was reduced by short hairpin RNA (shWASP cells). Many of the defects associated with the loss of WASP, such as podosome-dependent matrix degradation and chemotaxis were corrected when N-WASP was expressed at equimolar level to that of the wild-type WASP. Furthermore, the ability of N-WASP to partially compensate for the loss of WASP may be physiologically relevant since activated murine WASP-deficient peritoneal macrophages, which show enhanced N-WASP expression, also show an increase in matrix degradation. Our study suggests that expression levels of WASP and N-WASP may influence their roles in actin cytoskeleton rearrangement and shed light to the complex intertwining roles WASP and N-WASP play in macrophages.

Differential involvement of destrin and cofilin-1 in the control of invasive properties of Isreco1 human colon cancer cells.

Actin depolymerizing factor (ADF)/cofilin family proteins are key regulators of actin filament turnover and cytoskeleton reorganization. The role of cofilin-1 in cell motility has been demonstrated in several cell types but remained poorly documented in the case of colon cancer. In addition, the putative function of destrin (also known as ADF) had not been explored in this context despite the fact that it is expressed in all colon cancer cell lines examined. We were therefore prompted to evaluate the respective contributions of these proteins to the invasive properties of the human colon cancer Isreco1 cell line, which expresses a comparatively high destrin/cofilin ratio. Reduction of cofilin-1 or destrin expression in Isreco1 cells using RNA interference led to an increase of the number of multinucleated cells and altered polarized lamellipodium protrusion and distribution of paxillin-containing adhesions. Both cofilin-1 and destrin silencing enhanced cell adhesion to extracellular matrix components. However, only destrin appeared to be required for cell migration on collagen I and for cell invasion through Matrigel in response to the proinvasive neuroendocrine peptide bombesin. This differential functional involvement was supported by a destrin-dependent, cofilin-independent phosphorylation of p130Crk-associated substrate (p130Cas) upon cell adhesion to collagen I or Matrigel. Taken together, our results suggest that destrin is a significant regulator of various processes important for invasive phenotype of human colon cancer Isreco1 cells whereas cofilin-1 may be involved in only a subset of them.

Bombesin stimulates invasion and migration of Isreco1 colon carcinoma cells in a Rho-dependent manner.

The membrane receptor for the neuropeptide bombesin/gastrin-releasing peptide (GRP) is expressed by a large fraction of human colorectal carcinoma cells. We reported previously a stimulation of cell adhesion and lamellipodia formation by the neuropeptide bombesin in the human, bombesin/GRP receptor-expressing, Isreco1 colorectal cancer cell line (J. C. Saurin et al., Cancer Res., 59: 962-967, 1999). Using invasion and motility assays, we demonstrate in this report that bombesin can both enhance the invasive capacity of Isreco1 cells in a dose-dependent manner (maximal effect at 1 nM) and stimulate the closure of wounds performed on confluent Isreco1 cells. These effects were reversed fully by the specific bombesin/GRP receptor antagonist D-Phe(6)-Bn(6-13)OMe used at 1 micro M. MMP-9 and urokinase-type plasminogen activator were expressed by Isreco1 cells, and bombesin did not significantly alter their level of secretion. Interestingly, exoenzyme C3 (10 micro g/ml) decreased cell invasiveness induced by bombesin by 70% and completely inhibited the migration of Isreco1 cells. Similarly, the Rho-kinase inhibitor Y-27632 dose-dependently reduced the effect of bombesin on cell invasion. Moreover, pull-down assays for GTP-bound RhoA demonstrated that bombesin was able to activate the small G-protein in Isreco1 cells. These results show that the neuropeptide bombesin is able to modulate invasiveness of Isreco1 colorectal carcinoma cells in vitro through a Rho-dependent pathway, leading to an increase in cell locomotion without a significant effect on tumor-cell associated proteolytic activity. These findings indicate that bombesin/GRP receptor expression may contribute to the cellular events that are critical for invasion/migration of colorectal carcinoma cells.

The mechanism of CSF-1-induced Wiskott-Aldrich syndrome protein activation in vivo: a role for phosphatidylinositol 3-kinase and Cdc42.

A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.

Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

CX3CL1 (fractalkine), the only member of the delta subclass of chemokines, is a known chemotactic factor for monocytes/macrophages as well as NK cells and T lymphocytes. In several pathologies, excessive production of CX3CL1 at specific sites leads primarily to monocyte/macrophage recruitment, which causes tissue and vascular damage. Despite their clinical relevance, the mechanisms underlying monocyte/macrophage chemotaxis to CX3CL1 remain poorly documented. The present report addresses this issue and identifies cell signaling crucial for this process. Using the murine monocyte/macrophage RAW cell line, we show that CX3CL1 treatment elicits a rapid and transient increase in F-actin and the formation of F-actin-enriched cell protrusions. CX3CL1 also triggers tyrosine phosphorylation of proteins localized in those protrusions. The protein tyrosine kinase Syk is activated upon CX3CL1 treatment, and reduction of Syk expression using RNA-mediated interference results in a specific and massive impairment of RAW cell migration to CX3CL1. Similar results are obtained using the Syk inhibitor, piceatannol. Cells with reduced Syk expression also exhibit a major defect in CX3CL1-induced cytoskeletal remodeling. These data suggest that in monocytes/macrophages, Syk is essential for proper reorganization of the actin cytoskeleton in response to CX3CL1 and is therefore required for cell chemotaxis to CX3CL1.

A WAVE2-Abi1 complex mediates CSF-1-induced F-actin-rich membrane protrusions and migration in macrophages.

Colony-stimulating factor 1 (CSF-1) is an important physiological chemoattractant for macrophages. The mechanisms by which CSF-1 elicits the formation of filamentous actin (F-actin)-rich membrane protrusions and induces macrophage migration are not fully understood. In particular, very little is known regarding the contribution of the different members of the Wiskott-Aldrich Syndrome protein (WASP) family of actin regulators in response to CSF-1. Although a role for WASP itself in macrophage chemotaxis has been previously identified, no data was available regarding the function of WASP family verprolin-homologous (WAVE) proteins in this cell type. We found that WAVE2 was the predominant isoform to be expressed in primary macrophages and in cells derived from the murine monocyte/macrophage RAW264.7 cell line (RAW/LR5). CSF-1 treatment of macrophages resulted in WAVE2 accumulation in F-actin-rich protrusions induced by CSF-1. Inhibition of WAVE2 function by expressing a dominant-negative mutant or introducing anti-WAVE2 antibodies in RAW/LR5 cells, as well as reduction of endogenous WAVE2 expression by RNA-mediated interference (RNAi), resulted in a significant reduction of CSF-1-elicited F-actin protrusions. WAVE2 was found in a protein complex together with Abelson kinase interactor 1 (Abi1) in resting or stimulated cells. Both WAVE2 and Abi1 were recruited to and necessary for the formation of F-actin protrusions in response to CSF-1. Reducing the levels of WAVE2, directly or by targeting Abi1, resulted in an impaired cell migration to CSF-1. Altogether these data identify a WAVE2-Abi1 complex crucial for the normal actin cytoskeleton reorganization and migration of macrophages in response to CSF-1.

Co-requirement of cyclic AMP- and calcium-dependent protein kinases for transcriptional activation of cholecystokinin gene by protein hydrolysates.

Little is known about the mechanisms by which protein-derived nutrients regulate hormone gene expression in the intestine. We have previously reported that protein hydrolysates (i.e. peptones), which are representative of the protein fraction in the lumen, increased cholecystokinin (CCK) gene transcription in the STC-1 enteroendocrine cell line. In the present work, we examined the intracellular events evoked by peptones to stimulate CCK gene transcription. In STC-1 cells, peptones stimulated cyclic AMP production and protein kinase A (PKA) activity. This was associated with a nuclear translocation of the PKA catalytic subunit and with a PKA-dependent phosphorylation of the CRE-binding protein (CREB) at Ser(133). Using transient transfection experiments and reporter luciferase assays, we show that peptone-stimulated transcriptional activity of the CCK gene promoter was significantly decreased when the PKA pathway was inhibited. Furthermore, the intracellular calcium chelator 1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetra(acetoxymethyl)ester completely inhibited peptone-induced stimulation of the CCK gene promoter activity, phosphorylation of CREB, and PKA activity. Peptones increased, in a calcium-dependent manner, the phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and the MEK inhibitor PD98059 decreased the peptone-induced stimulation of CCK gene promoter activity. This stimulation was also reduced by 30% in the presence of the calcium/calmodulin-dependent protein kinase (CaMK) inhibitor KN-93. Total inhibition was obtained when the PKA, ERK, and CaMK pathways were simultaneously blocked with appropriate inhibitors to these pathways. These results demonstrate the simultaneous involvement of cAMP- and calcium-dependent protein kinases in the stimulation of intestinal CCK gene transcription by protein-derived nutrients.