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. 2005 Jan;25(1):60-5.
doi: 10.1128/MCB.25.1.60-65.2005.

MEKK1 transduces activin signals in keratinocytes to induce actin stress fiber formation and migration

Lin Zhang  1 , Maoxian Deng, Ranjani Parthasarathy, Lei Wang, Maureen Mongan, Jeffery D Molkentin, Yi Zheng, Ying Xia
Affiliations

MEKK1 transduces activin signals in keratinocytes to induce actin stress fiber formation and migration

Lin Zhang et al. Mol Cell Biol. 2005 Jan.

Abstract

Activins and other members of the transforming growth factor beta family play a critical role in morphological changes of the epidermis that require epithelial cell movement. We investigated the molecular pathways in the transmission of activin signals that lead to actin reorganization and epithelial cell migration. We found that activins cause the activation of RhoA but not of Rac and CDC42, leading to MEKK1-dependent phosphorylation of JNK and transcription factor c-Jun. Through a RhoA-independent mechanism, the activins also induce p38 activity in keratinocytes from wild-type but not from MEKK1-deficient mice. Although neither pathway is dependent on Smad activation, the MEKK1-mediated JNK and p38 activities are both essential for activin-stimulated and transcription-dependent keratinocyte migration. Only JNK is involved in transcription-independent actin stress fiber formation, which needs also the activity of ROCK. Because ROCK is required for JNK activation by RhoA and its overexpression leads to MEKK1 activation, we propose a RhoA-ROCK-MEKK1-JNK pathway and a MEKK1-p38 pathway as Smad-independent mechanisms in the transmission of activin signals. Together, these pathways lead to the control of actin cytoskeleton reorganization and epithelial cell migration, contributing to the physiologic and pathological effects of activins on epithelial morphogenesis.

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Figures

FIG. 1.
FIG. 1.
Activin-induced RhoA activation is required for actin stress fiber formation. GTP-bound and total RhoA (A) and Rac (B) were measured in keratinocytes treated with serum (10%), TGFα (10 ng/ml), or activin B (5 ng/ml) at the indicated doses for 10 min. Cell lysates were used to measure the levels of GTP-RhoA and GTP-Rac by GTP pull-down assay, and total RhoA and Rac1 was evaluated by Western blot analysis. The ratio of GTP-bound versus total GTPase is indicated. (C) Retroviruses carrying a GFP gene and genes for the enzyme-inactive mutant RhoA(N19), Rac(N17), and CDC42(N17) were used to infect wild-type keratinocytes. At 24 h after infection, the cells were treated with activin B (5 ng/ml) for 1 h. The cells were stained with rhodamine-phalloidin for F-actin and with DAPI for nuclei, and the virus-infected cells were identified by GFP fluorescence.
FIG. 2.
FIG. 2.
RhoA-stimulated actin stress fiber formation requires MEKK1. (A) Wild-type and Mekk1ΔKD/ΔKD keratinocytes were infected by retrovirus containing GFP and RhoA mutants as indicated. (B) Wild-type keratinocytes were infected with retroviruses containing the indicated RhoA mutants and adenoviruses that express constitutive-active (WT) or kinase-inactive (KM) MEKK1. The cells were stained for F-actin at 24 to 48 h after infection as described before.
FIG. 3.
FIG. 3.
RhoA induces MEKK1-dependent activation of JNK and c-Jun. Wild-type and Mekk1ΔKD/ΔKD keratinocytes were infected by retroviruses for GFP and various mutants of RhoA, Rac1, or CDC42, as indicated. (A) Cell lysates were analyzed for the phosphorylated isoforms and total JNK, p38, and ERK by Western blotting. (B) Cells were subjected to immunostaining with a polyclonal antibody against p-c-Jun, followed by rhodamine-anti-rabbit antibody and DAPI. Phosphorylated c-Jun, GFP, and DAPI were visualized by fluorescence microscopy. (C) A summary of the RhoA effector mutants in ROCK binding (19), induction of the MEKK1-dependent JNK phosphorylation, and actin stress fiber formation.
FIG. 4.
FIG. 4.
Activin B-induced, RhoA- and MEKK1-dependent JNK activity is essential, but p38 and protein syntheses are not required, for actin stress fiber formation. (A) Wild-type and Mekk1ΔKD/ΔKD keratinocytes were treated with activin B for the indicated times, and cell lysates were analyzed for the phosphorylated isoforms and total JNK, p38, and ERK by Western blotting. (B) Wild-type keratinocytes were infected with retroviruses containing empty vector or RhoA(N19). The cells were treated with activin B for 10 min before being harvested for Western blot analyses, as described above. (C) Wild-type keratinocytes were pretreated with chemical inhibitors as indicated, followed by stimulation with activin B and TGFα for 1 h. Polymerized actins were detected by rhodamine-phalloidin staining, and nuclei were identified by DAPI staining. SP, JNK inhibitor SP600125; SB, p38 inhibitor SB202190; CHX, protein synthesis inhibitor CHX.
FIG. 5.
FIG. 5.
JNK, but not Smad, activation is essential and sufficient for inducing actin stress fiber formation. (A and B) Wild-type keratinocytes were pretreated with various chemical inhibitors as indicated, followed by infection with retroviruses for RhoA(L63) (A) or adenoviruses for MEKK1(WT) (B). F-actins were detected by rhodamine-phalloidin staining, RhoA-positive cells were identified by using GFP, and nuclei were visualized by DAPI staining. (C) Cells infected by adenoviruses for HA-MEKK1(WT), HA-MEKK1(KM), active MKK6, and MKK7 were lysed. Cell lysates were analyzed by Western blotting for the expression of MEKK1 with anti-HA, of MKK6 with anti-MKK6, and of MKK7 with anti-pJNK and anti-JNK. (D) Some of the adenovirus-infected cells were treated with activin B for 1 h before fixation and immunostaining with FITC-phalloidin and α-Smad4, which was recognized by rhodamine-anti-rabbit immunoglobulin G. (E) Cells infected by retroviruses of RhoA(L63) and RhoA(N19) were recognized by GFP and were subjected to immunostaining with α-Smad4, as described above.
FIG. 6.
FIG. 6.
ROCK is required for actin stress fiber formation and the activation of the MEKK1-JNK pathway. (A) Wild-type andMekk1ΔKD/ΔKD keratinoyctes were infected by retroviruses of ROCK I in the presence or absence of the MAPK inhibitors, as indicated. (B) Wild-type keratinocytes were pretreated with Y27632 (10 μM), followed by infection with retroviruses expressing active RhoA(L63) or adenoviruses for active MEKK1(WT) and MKK7. Cells were analyzed for F-actin by using rhodamine-phalloidin, and nuclei were identified by DAPI staining. RhoA-positive cells were identified by using GFP as described before. (C) Cells with or without pretreatment with Y27632 were infected by retroviruses expressing active RhoA(L63) or adenoviruses for active MEKK1(WT). Cell lysates were analyzed by Western blotting for phosphorylated and total JNK. (D) Lysates from keratinocytes infected by ROCK I were subjected to immunocomplex kinase assay by using anti-MEKK1 for precipitation and GST-JNKK1 as a substrate in the presence of [γ-32P]ATP.
FIG. 7.
FIG. 7.
Both JNK and p38 are required for keratinocyte migration. (A) Primary mouse keratinocytes were used for an in vitro wound-healing assay. Cells were treated by activin B (10 ng/ml) or TGFα (5 ng/ml) in the presence of absence of various inhibitors at 5 μM. Pictures were taken at 0, 12, 24, and 36 h after wounding, and only the 24 h photos are shown. SP, JNK inhibitor SP600125; SB, p38 inhibitor SB202190; PD, ERK inhibitor PD98059; CHX, protein synthesis inhibitor CHX. (B) Model of molecular pathways by which activin signals induce actin stress fiber formation and keratinocyte migration. The activins cause activation of RhoA, which in turn induces the ROCK-dependent MEKK1-JNK pathway that leads to a transcription-independent actin stress fiber formation. ROCK, in regulating MLCK, might have a role independent of the MEKK1-JNK pathway in the control of actin cytoskeleton. The JNK might regulate actin stress fiber formation through phosphorylation of paxillin. Apart from actin stress fiber formation, this pathway also results in the induction of c-Jun phosphorylation, which might lead to transcription-dependent events. The activin signals transduce also through a RhoA-independent MEKK1-p38 pathway that is critical for the transcription-dependent migration. Neither pathway is connected to Smad activation.
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