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A negative modulatory role for rho and rho-associated kinase signaling in delamination of neural crest cells.

Groysman M, Shoval I, Kalcheim C - Neural Dev (2008)

Bottom Line: Reciprocally, activation of endogenous Rho by lysophosphatidic acid inhibited emigration while enhancing the above.In the latter condition, cells emigrated while arrested at G1.Conversely, BMP4 was unable to rescue cell emigration when endogenous Rho activity was enhanced by lysophosphatidic acid.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. mayagr@ekmd.huji.ac.il

ABSTRACT

Background: Neural crest progenitors arise as epithelial cells and then undergo a process of epithelial to mesenchymal transition that precedes the generation of cellular motility and subsequent migration. We aim at understanding the underlying molecular network. Along this line, possible roles of Rho GTPases that act as molecular switches to control a variety of signal transduction pathways remain virtually unexplored, as are putative interactions between Rho proteins and additional known components of this cascade.

Results: We investigated the role of Rho/Rock signaling in neural crest delamination. Active RhoA and RhoB are expressed in the membrane of epithelial progenitors and are downregulated upon delamination. In vivo loss-of-function of RhoA or RhoB or of overall Rho signaling by C3 transferase enhanced and/or triggered premature crest delamination yet had no effect on cell specification. Consistently, treatment of explanted neural primordia with membrane-permeable C3 or with the Rock inhibitor Y27632 both accelerated and enhanced crest emigration without affecting cell proliferation. These treatments altered neural crest morphology by reducing stress fibers, focal adhesions and downregulating membrane-bound N-cadherin. Reciprocally, activation of endogenous Rho by lysophosphatidic acid inhibited emigration while enhancing the above. Since delamination is triggered by BMP and requires G1/S transition, we examined their relationship with Rho. Blocking Rho/Rock function rescued crest emigration upon treatment with noggin or with the G1/S inhibitor mimosine. In the latter condition, cells emigrated while arrested at G1. Conversely, BMP4 was unable to rescue cell emigration when endogenous Rho activity was enhanced by lysophosphatidic acid.

Conclusion: Rho-GTPases, through Rock, act downstream of BMP and of G1/S transition to negatively regulate crest delamination by modifying cytoskeleton assembly and intercellular adhesion.

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The role of Rho/Rock signaling in the molecular network underlying neural crest (NC) delamination. Opposite the segmental plate mesoderm, high levels of noggin result in low bone morphogenetic protein (BMP) activity, no Wnt1 transcription, low cyclin D1 in dorsal neural tube (NT) and no NC cells emigrating from the caudal NT. N-cadherin at this stage is expressed in the dorsal NT where it contributes to maintaining low cyclin D1 and lack of NC emigration. Rho activity through Rock helps maintaining membrane-bound N-cadherin and keeps a stable F-actin cytoskeleton. Together with the previous, direct N-cadherin-F-actin interactions contribute to the maintenance of epithelial premigratory NC. With ongoing development, opposite mature epithelial and dissociating somites, a factor emitted by the dorsomedial portion of the paraxial mesoderm inhibits noggin transcription in the NT, thereby relieving BMP activity. BMP4 in turn triggers Wnt1 transcription. Canonical Wnt signaling positively modulates transcription of cyclin D1, G1/S transition and NC cell delamination. In parallel, BMP4 via ADAM10 promotes N-cadherin cleavage into soluble CTF2. CTF2 may act in at least two ways, by upregulating β-catenin transcription and by binding β-catenin protein; we proposed that the complex translocates into the cell nucleus where transcription of target genes such as cyclin D1, followed by G1/S transition and epithelial-to-mesenchymal transition (EMT) of NC are stimulated. Hence, BMP activity transforms N-cadherin into a stimulatory signal (for details, see [31]. Concomitant with delamination, membrane-bound Rho proteins are downregulated, suggesting reduced activities of both Rho and Rock proteins; consequently, a dynamic turnover of stress fibers is made possible and N-cadherin association to the membrane is relieved. Altogether, these processes are compatible with generation of cellular movement downstream of the G1/S transition phase.
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Figure 11: The role of Rho/Rock signaling in the molecular network underlying neural crest (NC) delamination. Opposite the segmental plate mesoderm, high levels of noggin result in low bone morphogenetic protein (BMP) activity, no Wnt1 transcription, low cyclin D1 in dorsal neural tube (NT) and no NC cells emigrating from the caudal NT. N-cadherin at this stage is expressed in the dorsal NT where it contributes to maintaining low cyclin D1 and lack of NC emigration. Rho activity through Rock helps maintaining membrane-bound N-cadherin and keeps a stable F-actin cytoskeleton. Together with the previous, direct N-cadherin-F-actin interactions contribute to the maintenance of epithelial premigratory NC. With ongoing development, opposite mature epithelial and dissociating somites, a factor emitted by the dorsomedial portion of the paraxial mesoderm inhibits noggin transcription in the NT, thereby relieving BMP activity. BMP4 in turn triggers Wnt1 transcription. Canonical Wnt signaling positively modulates transcription of cyclin D1, G1/S transition and NC cell delamination. In parallel, BMP4 via ADAM10 promotes N-cadherin cleavage into soluble CTF2. CTF2 may act in at least two ways, by upregulating β-catenin transcription and by binding β-catenin protein; we proposed that the complex translocates into the cell nucleus where transcription of target genes such as cyclin D1, followed by G1/S transition and epithelial-to-mesenchymal transition (EMT) of NC are stimulated. Hence, BMP activity transforms N-cadherin into a stimulatory signal (for details, see [31]. Concomitant with delamination, membrane-bound Rho proteins are downregulated, suggesting reduced activities of both Rho and Rock proteins; consequently, a dynamic turnover of stress fibers is made possible and N-cadherin association to the membrane is relieved. Altogether, these processes are compatible with generation of cellular movement downstream of the G1/S transition phase.

Mentions: We show that the effects on NC emigration caused by changing the levels of Rho activity cannot be explained by altered cell proliferation, cell survival or cell specification. Thus, the enhanced cell emigration observed in Rho loss of function experiments may reflect premature depletion of the subset of transfected NC progenitors due to an earlier than normal loss of cell adhesion and cytoskeletal stability that characterize the epithelial state. Indeed, enhanced NC delamination produced by inhibiting Rho/Rock is accompanied by a substantial loss of actin stress fibers and focal adhesions. In addition, we demonstrate that membrane-bound N-cadherin is lost under these conditions, even if its normal proteolytic degradation is inhibited [31], and reciprocally, when preventing N-cadherin degradation, stable stress fibers, a representation of Rho activity, are kept. Furthermore, when endogenous Rho is activated by LPA, the observed inhibition of cell delamination is associated with maintenance of membrane N-cadherin in ovo and explants. This confirms that in the NC, Rho proteins together with N-cadherin are negative effectors of the generation of cellular movement. Consistent with our results, cooperation between cadherins and the Rho-dependent actin cytoskeleton were shown to control many aspects of epithelial biogenesis and maintenance [75-83]. On the one hand, cadherin-mediated adhesion is both necessary and sufficient for small GTPase activation, and on the other hand, sustained Rho activity is required for N-cadherin mediated adhesion, likely through maintenance of cytoskeletal stability [83,84]. Based on these observations, we propose that in the dorsal NT at premigratory levels of the axis, where membrane-associated N-cadherin is strongly expressed, Rho activity is maximal, thus contributing to preservation of the epithelial state of presumptive NC cells (Figure 11). When noggin activity is downregulated and BMP is consequently activated, N-cadherin is proteolytically degraded in the dorsal NT via a BMP and ADAM10-dependent mechanism [31]. Loss of membrane bound N-cadherin could signal a reduction in Rho activity via modifications of the actin cytoskeleton. Alternatively, or in addition, BMP, a key regulator of NC EMT, could contribute to RhoA degradation [64]. In this context, it is important to emphasize that BMP was shown to be necessary for inducing and maintaining transcription of RhoB mRNA in the dorsal NT [20,40]. However, monitoring mRNA expression is not a predictive factor for Rho activity as C3 transferase, despite abolishing Rho function, did not affect levels of RhoB mRNA (MG and CK, unpublished); hence, the regulation of Rho transcription and protein activity are separable events [85]. Additional levels of regulation should be considered as well; for instance, RhoB protein was shown to be stabilized against proteolytic degradation by transforming growth factor-β; in turn, RhoB antagonized transforming growth factor-β-dependent transcriptional activation [86]. Hence, the possibility should be considered that, in our system as well, stabilized RhoB antagonizes BMP-dependent EMT of NC cells.


A negative modulatory role for rho and rho-associated kinase signaling in delamination of neural crest cells.

Groysman M, Shoval I, Kalcheim C - Neural Dev (2008)

The role of Rho/Rock signaling in the molecular network underlying neural crest (NC) delamination. Opposite the segmental plate mesoderm, high levels of noggin result in low bone morphogenetic protein (BMP) activity, no Wnt1 transcription, low cyclin D1 in dorsal neural tube (NT) and no NC cells emigrating from the caudal NT. N-cadherin at this stage is expressed in the dorsal NT where it contributes to maintaining low cyclin D1 and lack of NC emigration. Rho activity through Rock helps maintaining membrane-bound N-cadherin and keeps a stable F-actin cytoskeleton. Together with the previous, direct N-cadherin-F-actin interactions contribute to the maintenance of epithelial premigratory NC. With ongoing development, opposite mature epithelial and dissociating somites, a factor emitted by the dorsomedial portion of the paraxial mesoderm inhibits noggin transcription in the NT, thereby relieving BMP activity. BMP4 in turn triggers Wnt1 transcription. Canonical Wnt signaling positively modulates transcription of cyclin D1, G1/S transition and NC cell delamination. In parallel, BMP4 via ADAM10 promotes N-cadherin cleavage into soluble CTF2. CTF2 may act in at least two ways, by upregulating β-catenin transcription and by binding β-catenin protein; we proposed that the complex translocates into the cell nucleus where transcription of target genes such as cyclin D1, followed by G1/S transition and epithelial-to-mesenchymal transition (EMT) of NC are stimulated. Hence, BMP activity transforms N-cadherin into a stimulatory signal (for details, see [31]. Concomitant with delamination, membrane-bound Rho proteins are downregulated, suggesting reduced activities of both Rho and Rock proteins; consequently, a dynamic turnover of stress fibers is made possible and N-cadherin association to the membrane is relieved. Altogether, these processes are compatible with generation of cellular movement downstream of the G1/S transition phase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577655&req=5

Figure 11: The role of Rho/Rock signaling in the molecular network underlying neural crest (NC) delamination. Opposite the segmental plate mesoderm, high levels of noggin result in low bone morphogenetic protein (BMP) activity, no Wnt1 transcription, low cyclin D1 in dorsal neural tube (NT) and no NC cells emigrating from the caudal NT. N-cadherin at this stage is expressed in the dorsal NT where it contributes to maintaining low cyclin D1 and lack of NC emigration. Rho activity through Rock helps maintaining membrane-bound N-cadherin and keeps a stable F-actin cytoskeleton. Together with the previous, direct N-cadherin-F-actin interactions contribute to the maintenance of epithelial premigratory NC. With ongoing development, opposite mature epithelial and dissociating somites, a factor emitted by the dorsomedial portion of the paraxial mesoderm inhibits noggin transcription in the NT, thereby relieving BMP activity. BMP4 in turn triggers Wnt1 transcription. Canonical Wnt signaling positively modulates transcription of cyclin D1, G1/S transition and NC cell delamination. In parallel, BMP4 via ADAM10 promotes N-cadherin cleavage into soluble CTF2. CTF2 may act in at least two ways, by upregulating β-catenin transcription and by binding β-catenin protein; we proposed that the complex translocates into the cell nucleus where transcription of target genes such as cyclin D1, followed by G1/S transition and epithelial-to-mesenchymal transition (EMT) of NC are stimulated. Hence, BMP activity transforms N-cadherin into a stimulatory signal (for details, see [31]. Concomitant with delamination, membrane-bound Rho proteins are downregulated, suggesting reduced activities of both Rho and Rock proteins; consequently, a dynamic turnover of stress fibers is made possible and N-cadherin association to the membrane is relieved. Altogether, these processes are compatible with generation of cellular movement downstream of the G1/S transition phase.
Mentions: We show that the effects on NC emigration caused by changing the levels of Rho activity cannot be explained by altered cell proliferation, cell survival or cell specification. Thus, the enhanced cell emigration observed in Rho loss of function experiments may reflect premature depletion of the subset of transfected NC progenitors due to an earlier than normal loss of cell adhesion and cytoskeletal stability that characterize the epithelial state. Indeed, enhanced NC delamination produced by inhibiting Rho/Rock is accompanied by a substantial loss of actin stress fibers and focal adhesions. In addition, we demonstrate that membrane-bound N-cadherin is lost under these conditions, even if its normal proteolytic degradation is inhibited [31], and reciprocally, when preventing N-cadherin degradation, stable stress fibers, a representation of Rho activity, are kept. Furthermore, when endogenous Rho is activated by LPA, the observed inhibition of cell delamination is associated with maintenance of membrane N-cadherin in ovo and explants. This confirms that in the NC, Rho proteins together with N-cadherin are negative effectors of the generation of cellular movement. Consistent with our results, cooperation between cadherins and the Rho-dependent actin cytoskeleton were shown to control many aspects of epithelial biogenesis and maintenance [75-83]. On the one hand, cadherin-mediated adhesion is both necessary and sufficient for small GTPase activation, and on the other hand, sustained Rho activity is required for N-cadherin mediated adhesion, likely through maintenance of cytoskeletal stability [83,84]. Based on these observations, we propose that in the dorsal NT at premigratory levels of the axis, where membrane-associated N-cadherin is strongly expressed, Rho activity is maximal, thus contributing to preservation of the epithelial state of presumptive NC cells (Figure 11). When noggin activity is downregulated and BMP is consequently activated, N-cadherin is proteolytically degraded in the dorsal NT via a BMP and ADAM10-dependent mechanism [31]. Loss of membrane bound N-cadherin could signal a reduction in Rho activity via modifications of the actin cytoskeleton. Alternatively, or in addition, BMP, a key regulator of NC EMT, could contribute to RhoA degradation [64]. In this context, it is important to emphasize that BMP was shown to be necessary for inducing and maintaining transcription of RhoB mRNA in the dorsal NT [20,40]. However, monitoring mRNA expression is not a predictive factor for Rho activity as C3 transferase, despite abolishing Rho function, did not affect levels of RhoB mRNA (MG and CK, unpublished); hence, the regulation of Rho transcription and protein activity are separable events [85]. Additional levels of regulation should be considered as well; for instance, RhoB protein was shown to be stabilized against proteolytic degradation by transforming growth factor-β; in turn, RhoB antagonized transforming growth factor-β-dependent transcriptional activation [86]. Hence, the possibility should be considered that, in our system as well, stabilized RhoB antagonizes BMP-dependent EMT of NC cells.

Bottom Line: Reciprocally, activation of endogenous Rho by lysophosphatidic acid inhibited emigration while enhancing the above.In the latter condition, cells emigrated while arrested at G1.Conversely, BMP4 was unable to rescue cell emigration when endogenous Rho activity was enhanced by lysophosphatidic acid.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. mayagr@ekmd.huji.ac.il

ABSTRACT

Background: Neural crest progenitors arise as epithelial cells and then undergo a process of epithelial to mesenchymal transition that precedes the generation of cellular motility and subsequent migration. We aim at understanding the underlying molecular network. Along this line, possible roles of Rho GTPases that act as molecular switches to control a variety of signal transduction pathways remain virtually unexplored, as are putative interactions between Rho proteins and additional known components of this cascade.

Results: We investigated the role of Rho/Rock signaling in neural crest delamination. Active RhoA and RhoB are expressed in the membrane of epithelial progenitors and are downregulated upon delamination. In vivo loss-of-function of RhoA or RhoB or of overall Rho signaling by C3 transferase enhanced and/or triggered premature crest delamination yet had no effect on cell specification. Consistently, treatment of explanted neural primordia with membrane-permeable C3 or with the Rock inhibitor Y27632 both accelerated and enhanced crest emigration without affecting cell proliferation. These treatments altered neural crest morphology by reducing stress fibers, focal adhesions and downregulating membrane-bound N-cadherin. Reciprocally, activation of endogenous Rho by lysophosphatidic acid inhibited emigration while enhancing the above. Since delamination is triggered by BMP and requires G1/S transition, we examined their relationship with Rho. Blocking Rho/Rock function rescued crest emigration upon treatment with noggin or with the G1/S inhibitor mimosine. In the latter condition, cells emigrated while arrested at G1. Conversely, BMP4 was unable to rescue cell emigration when endogenous Rho activity was enhanced by lysophosphatidic acid.

Conclusion: Rho-GTPases, through Rock, act downstream of BMP and of G1/S transition to negatively regulate crest delamination by modifying cytoskeleton assembly and intercellular adhesion.

Show MeSH
Related in: MedlinePlus