C-Jun N-terminal kinase (JNK) mediates Wnt5a-induced cell motility dependent or independent of RhoA pathway in human dental papilla cells

Abstract

Wnt5a plays an essential role in tissue development by regulating cell migration, though the molecular mechanisms are still not fully understood. Our study investigated the pathways involved in Wnt5a-dependent cell motility during the formation of dentin and pulp. Over-expression of Wnt5a promoted cell adhesion and formation of focal adhesion complexes (FACs) in human dental papilla cells (hDPCs), while inhibiting cell migration. Instead of activating the canonical Wnt signal pathway in hDPCs, Wnt5a stimulation induced activation of the JNK signal in a RhoA-dependent or independent manner. Inhibiting JNK abrogated Wnt5a-induced FACs formation but not cytoskeletal rearrangement. Both dominant negative RhoA (RhoA T19N) and constitutively active RhoA mutants (RhoA Q63L) blocked the Wnt5a-dependent changes in hDPCs adhesion, migration and cytoskeletal rearrangement here too, with the exception of the formation of FACs. Taken together, our study suggested that RhoA and JNK signaling have roles in mediating Wnt5a-dependent adhesion and migration in hDPCs, and the Wnt5a/JNK pathway acts both dependently and independently of the RhoA pathway.

Figure 1. Wnt5a promotes the adhesion but inhibits the migration of hDPCs.

A: A total of 25,000 cells were seeded for the indicated times and nonadherent cells were rinsed off. Adherent cells were stained with crystal violet and were analyzed spectrophotometrically. The number of adherent cells is shown as mean±SD from three independent experiments, with the number of DMEM-treated cells set as 100%. B: Confluent monolayers of hDPCs were scratched with a pipette tip and cultured in different medium containing 5% FBS for 12 hr. The relative migration distance of the wound edge was shown as mean±SD of three independent experiments, with the migration distance of DMEM-treated cells set as 100%. Bars, 100 µm. C: HDPCs were plated on glass coverslips coated with type I collagen and cultured with different medium for the indicated times. For FACs immunostaining, anti-vinculin antibody was used, and for F-actin staining, rhodamine-phalloidin was used, arrowheads mark FACs. Bars,10 µm. D: Confluent hDPCs were incubated with Wnt5a for the indicated times and Western analyses were used to detect the expression of vinculin, phospho-paxillin, phospho-MLC, with GAPDH, total-paxillin and total-MLC as loading control. The relative protein expression at 0 min is designated 1.0. *p < 0.05, n=3.

Figure 2. Wnt5a has no effect on β-catenin expression or translocation in hDPCs.

A: Western analyses of β-catenin in cytosolic or nuclear fractions of cells cultured in Wnt5a CM for indicated times. Cytosolic and nuclear signals were normalized to GAPDH and H3, respectively. The relative expression of β-catenin at 0min is designated 1.0. B: Immunofluorescence microscopy of hDPCs following culture with rhWnt5a or Wnt5a CM for 1 hr. β-catenin signal is in green. Bars,30 µm. C: Wnt5a up-regulates the expression of GTP-RhoA and phospho-JNK. RhoA activity stimulated by Wnt5a was detected by GST-Pull down assay at the indicated times, the expression of total RhoA, phospho-JNK, and total-JNK was detected by Western blot analysis. The relative protein expression at 0min is designated 1.0. *p < 0.05, n=3.

Figure 3. The role of the JNK pathway in Wnt5a-dependent adhesion, migration and formation of FACs.

A: HDPCs were pretreated with 10 µM or 30 µM SP600125 for 30 min and the levels of phospho-JNK and total JNK were measured by Western blot analysis. The relative protein expression without SP600125 treatment is designated 1.0. B ,C: Cell adhesion and wound healing assays were performed as in Figure 1, but the cells were hDPCs which were pretreated with 30 µm SP600125 for 30 min, and the observed time in the wound healing assay was 16 hr. Bars, 100 µm. D: Vinculin immunostaining and phalloidin staining were performed at 15min as in Figure 1C, but the hDPCs were pretreated with 30µM SP600125 for 30 min before being seeded onto glass slides. Arrowheads mark FACs. The number of FACs and the relative fluorescence were analyzed as in Figure 1C. Bars,10 µm. E: Pretreatment with 30 µM SP600125 for 30 min, hDPCs were incubated with Wnt5a CM for the indicated times, the cell lysates were collected and immunoblotted with antibodies to phospho-MLC, phospho-paxillin, total-paxillin, total-MLC and vinculin. The promotion of phospho-paxillin by Wnt5a CM was delayed until after 60 min, but no changes were seen in the expression of phospho-MLC. *p < 0.05, n = 3.

Figure 4. RhoA signaling contributed to the Wnt5a-dependent adhesion, migration changes and formation of FACs.

A: After infection with RhoA mutant adenoviruses, hDPCs were cultured with GFP CM or Wnt5a CM and cell adhesion assays were performed, as in Figure 1A. B: Confluent hDPCs infected with RhoA mutant adenoviruses for 48 hr were scratched and cultured with GFP CM or Wnt5a CM for 20 hr to observe the effect of Wnt5a CM on the cell. Bars,100 µm. C: Vinculin immunostaining and phalloidin staining were performed as in Figure 1C, but the hDPCs were infected with different RhoA mutant adenoviruses and the secondary antibody was Alexa 546-labeled. Arrowheads mark FACs. The number of FACs and the relative fluorescence were analyzed as if Figure 1C. Bars, 10 µm. D: After infection with RhoA T19N or RhoA Q63L adenoviruses for 48 hr, hDPCs were incubated with Wnt5a CM for the indicated times and collected for protein extraction. Western analyses of phospho-MLC, phospho-paxillin, total-paxillin, total-MLC and vinculin in hDPCs. a p < 0.05, n = 3, compared with RhoA WT at the same time point. *p < 0.05, n = 3.

Figure 5. Wnt5a activated JNK signaling is dependent and independent of RhoA signaling.

A: Lysates from hDPCs were obtained following transfection with adenoviral vectors encoding GFP, RhoA WT, RhoA T19N and RhoA Q63L for 48 hr and the levels of phospho-JNK and total-JNK were measured by Western blot analysis. The normalized amount of phospho-JNK in GFP adenovirus infected hDPCs is designated 1.0. B: HDPCs infected with adenoviruses encoding the RhoA mutant for 48 hr were cultured with Wnt5a CM and the cell lysates were obtained at the indicated times, with the level of phospho-JNK and total-JNK measured by Western analyses. The relative amount of phospho-JNK is normalized to the total amount, at 0min, is designated 1.0. *p < 0.05, n=3.

Figure 6. The role of the RhoA and JNK pathways in Wnt5a-dependent hDPC motility.