Figure 4.
Figure 4. Regulation of RhoA by STAT3 determines actomyosin tonus and migration in astrocytes. (A) Central stress fibers that formed 15 h after seeding onto laminin are stronger in STAT3-CKO astrocytes than in WT astrocytes. Bars, 20 µm. (B) These ventral stress fibers are sensitive to inhibition of ROCK in contrast to the peripheral actin ring, which is resistant. Bars, 20 µm. (C) Collagen gel contraction assay revealed an elevated actomyosin tonus in STAT3-CKO astrocytes, whereas contraction in response to TGF-α is not modified when compared with WT cells (data shown from three independent experiments in triplicate, one-way ANOVA followed by Bonferroni’s test). (D) RhoA is constitutively activated in STAT3-CKO astrocytes (n = 4 per group; Mann-Whitney U test; P = 0.0286). (E) Significant down-regulation of RhoA in STAT3-CKO astrocytes (Mann-Whitney U test, n = 7 and 9, in WT and STAT3-CKO, respectively; P = 0.0007). (F) Phosphorylation of MLC2 is increased in STAT3-CKO astrocytes in the usual culture conditions and after stimulation with human LIF (hLIF; G). The kinetics and amplitude of myosin light chain (MLC) phosphorylation are changed in STAT3-CKO astrocytes. Note the absence of induction of SOCS3 in response to hLIF in STAT3-CKO cells and that bands in (G) were from two separate gels that were processed in parallel. (H) In contrast to WT astrocytes, migration of STAT3-CKO astrocytes in the trans-well assay is insensitive to inhibition of RhoA-GTPase with 2 µg/ml of cell-permeable C3 exoenzyme (CT04). (I and J) Migration in the presence of two different ROCK inhibitors resulted in the same observation as in (H). (K) The activation level of STAT3 in astrocytes is not reduced upon ROCK inhibition. WT astrocytes were treated for 6 h with 20 µM Y27632. Treatment with MLC kinase inhibitor (L) or myosin II ATPase inhibitor (M) further showed that STAT3 is necessary for the RhoA-GTPase–dependent migration of astrocytes. (N) Phosphorylation of ezrin on Thr567 is selectively reduced in STAT3-CKO astrocytes, compared with WT astrocytes under the usual culture conditions (Mann-Whitney U test, P < 0.0001, n = 6 per group). (O) The level of GRK2 mRNA is unchanged in STAT3-CKO astrocytes versus WT astrocytes. (Normalization is made with GAPDH, Mann-Whitney U test, n = 5 per group). Ctrl, control; ns, not significant; Veh, vehicle. Error bars indicate ±SEM. *, P < 0.05; ***, P < 0.001.

Regulation of RhoA by STAT3 determines actomyosin tonus and migration in astrocytes. (A) Central stress fibers that formed 15 h after seeding onto laminin are stronger in STAT3-CKO astrocytes than in WT astrocytes. Bars, 20 µm. (B) These ventral stress fibers are sensitive to inhibition of ROCK in contrast to the peripheral actin ring, which is resistant. Bars, 20 µm. (C) Collagen gel contraction assay revealed an elevated actomyosin tonus in STAT3-CKO astrocytes, whereas contraction in response to TGF-α is not modified when compared with WT cells (data shown from three independent experiments in triplicate, one-way ANOVA followed by Bonferroni’s test). (D) RhoA is constitutively activated in STAT3-CKO astrocytes (n = 4 per group; Mann-Whitney U test; P = 0.0286). (E) Significant down-regulation of RhoA in STAT3-CKO astrocytes (Mann-Whitney U test, n = 7 and 9, in WT and STAT3-CKO, respectively; P = 0.0007). (F) Phosphorylation of MLC2 is increased in STAT3-CKO astrocytes in the usual culture conditions and after stimulation with human LIF (hLIF; G). The kinetics and amplitude of myosin light chain (MLC) phosphorylation are changed in STAT3-CKO astrocytes. Note the absence of induction of SOCS3 in response to hLIF in STAT3-CKO cells and that bands in (G) were from two separate gels that were processed in parallel. (H) In contrast to WT astrocytes, migration of STAT3-CKO astrocytes in the trans-well assay is insensitive to inhibition of RhoA-GTPase with 2 µg/ml of cell-permeable C3 exoenzyme (CT04). (I and J) Migration in the presence of two different ROCK inhibitors resulted in the same observation as in (H). (K) The activation level of STAT3 in astrocytes is not reduced upon ROCK inhibition. WT astrocytes were treated for 6 h with 20 µM Y27632. Treatment with MLC kinase inhibitor (L) or myosin II ATPase inhibitor (M) further showed that STAT3 is necessary for the RhoA-GTPase–dependent migration of astrocytes. (N) Phosphorylation of ezrin on Thr567 is selectively reduced in STAT3-CKO astrocytes, compared with WT astrocytes under the usual culture conditions (Mann-Whitney U test, P < 0.0001, n = 6 per group). (O) The level of GRK2 mRNA is unchanged in STAT3-CKO astrocytes versus WT astrocytes. (Normalization is made with GAPDH, Mann-Whitney U test, n = 5 per group). Ctrl, control; ns, not significant; Veh, vehicle. Error bars indicate ±SEM. *, P < 0.05; ***, P < 0.001.

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