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Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

Todorovic V, Koetsier JL, Godsel LM, Green KJ - Mol. Biol. Cell (2014)

Bottom Line: Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly.We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing.These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.

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Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC activator (PMA) and a RhoA inhibitor (C3) in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001 < p < 0.01, ***p < 0.001 (ANOVA, Bonferroni). (F) Cell–cell adhesion strength assay demonstrating recovery of adhesion strength in Pkp3-ablated cells treated by FSK and ISO, as well as disruption of the adhesion strength in PROP-treated cells. Bars represent mean ± SEM. ns, p > 0.05, ***p < 0.001 (ANOVA, Bonferroni).
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Figure 5: Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC activator (PMA) and a RhoA inhibitor (C3) in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001 < p < 0.01, ***p < 0.001 (ANOVA, Bonferroni). (F) Cell–cell adhesion strength assay demonstrating recovery of adhesion strength in Pkp3-ablated cells treated by FSK and ISO, as well as disruption of the adhesion strength in PROP-treated cells. Bars represent mean ± SEM. ns, p > 0.05, ***p < 0.001 (ANOVA, Bonferroni).

Mentions: We previously showed that PKCα activation and inhibition of sustained RhoA activity restored proper desmosome assembly and allowed for DP border localization in Pkp2-ablated cells (Bass-Zubek et al., 2008; Godsel et al., 2010). To test whether any of the aforementioned pathways are involved in Pkp3-dependent desmosome assembly, we treated control and Pkp2 and 3 KD keratinocytes with a PKC activator (phorbol 12-myristate 13-acetate [PMA]), a RhoA inhibitor (C3), and an adenylyl cyclase activator (forskolin [FSK]). Activation of PKC and RhoA inhibition restored normal DP localization in Pkp2 KD cells, as expected (Figure 5A). In contrast, DP cell–cell border localization was not rescued by these treatments in Pkp3 KD cells (Figure 5A and Supplemental Figure S2A). However, activation of cAMP production by FSK treatment (Supplemental Figure S2B) restored DP localization upon Pkp3 KD but had no effect on Pkp2 KD, illustrating that Pkp3 and Pkp2 modulate DP localization/trafficking via different pathways (Figure 5A). Two-day FSK treatment of organotypic epidermal cultures differentiated over a 6 d period also restored DP to its proper location in Pkp3 KD rafts (Figure 5B), demonstrating that adenylyl cyclase activation can still rescue the effects of Pkp3 ablation in differentiating keratinocytes and in the presence of Pkp1. To test whether inhibition of cAMP production could recapitulate the DP phenotype observed in Pkp3-silenced cells, we treated cells with the β-adrenergic receptor antagonist propranolol (PROP) and its agonist isoprenaline (ISO) as a control. As expected, treatment with ISO restored DP border localization in Pkp3 KD cells similarly to FSK, whereas treatment with PROP caused a disruption of DP border localization (Figure 5, C–E). At the same time, treatment of Pkp3 KD cells with either FSK or ISO completely restored cell–cell adhesion strength to detached monolayers, whereas treatment with PROP disrupted cell–cell adhesion to the level similar to that observed in Pkp3 KD (Figure 5F).


Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

Todorovic V, Koetsier JL, Godsel LM, Green KJ - Mol. Biol. Cell (2014)

Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC activator (PMA) and a RhoA inhibitor (C3) in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001 < p < 0.01, ***p < 0.001 (ANOVA, Bonferroni). (F) Cell–cell adhesion strength assay demonstrating recovery of adhesion strength in Pkp3-ablated cells treated by FSK and ISO, as well as disruption of the adhesion strength in PROP-treated cells. Bars represent mean ± SEM. ns, p > 0.05, ***p < 0.001 (ANOVA, Bonferroni).
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Figure 5: Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC activator (PMA) and a RhoA inhibitor (C3) in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001 < p < 0.01, ***p < 0.001 (ANOVA, Bonferroni). (F) Cell–cell adhesion strength assay demonstrating recovery of adhesion strength in Pkp3-ablated cells treated by FSK and ISO, as well as disruption of the adhesion strength in PROP-treated cells. Bars represent mean ± SEM. ns, p > 0.05, ***p < 0.001 (ANOVA, Bonferroni).
Mentions: We previously showed that PKCα activation and inhibition of sustained RhoA activity restored proper desmosome assembly and allowed for DP border localization in Pkp2-ablated cells (Bass-Zubek et al., 2008; Godsel et al., 2010). To test whether any of the aforementioned pathways are involved in Pkp3-dependent desmosome assembly, we treated control and Pkp2 and 3 KD keratinocytes with a PKC activator (phorbol 12-myristate 13-acetate [PMA]), a RhoA inhibitor (C3), and an adenylyl cyclase activator (forskolin [FSK]). Activation of PKC and RhoA inhibition restored normal DP localization in Pkp2 KD cells, as expected (Figure 5A). In contrast, DP cell–cell border localization was not rescued by these treatments in Pkp3 KD cells (Figure 5A and Supplemental Figure S2A). However, activation of cAMP production by FSK treatment (Supplemental Figure S2B) restored DP localization upon Pkp3 KD but had no effect on Pkp2 KD, illustrating that Pkp3 and Pkp2 modulate DP localization/trafficking via different pathways (Figure 5A). Two-day FSK treatment of organotypic epidermal cultures differentiated over a 6 d period also restored DP to its proper location in Pkp3 KD rafts (Figure 5B), demonstrating that adenylyl cyclase activation can still rescue the effects of Pkp3 ablation in differentiating keratinocytes and in the presence of Pkp1. To test whether inhibition of cAMP production could recapitulate the DP phenotype observed in Pkp3-silenced cells, we treated cells with the β-adrenergic receptor antagonist propranolol (PROP) and its agonist isoprenaline (ISO) as a control. As expected, treatment with ISO restored DP border localization in Pkp3 KD cells similarly to FSK, whereas treatment with PROP caused a disruption of DP border localization (Figure 5, C–E). At the same time, treatment of Pkp3 KD cells with either FSK or ISO completely restored cell–cell adhesion strength to detached monolayers, whereas treatment with PROP disrupted cell–cell adhesion to the level similar to that observed in Pkp3 KD (Figure 5F).

Bottom Line: Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly.We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing.These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.

Show MeSH
Related in: MedlinePlus