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Protein kinase C spatially and temporally regulates gap junctional communication during human wound repair via phosphorylation of connexin43 on serine368.

Richards TS, Dunn CA, Carter WG, Usui ML, Olerud JE, Lampe PD - J. Cell Biol. (2004)

Bottom Line: Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance.However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it).Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance. Examination of phosphoserine368 (pS368) in normal human skin tissue using a phosphorylation site-specific antibody showed relatively even distribution throughout the epidermal layers. However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it). Scratch wounding of primary human keratinocytes caused a protein kinase C (PKC)-dependent increase in pS368 in cells adjacent to the scratch, with a time course similar to that found in the wounds. Keratinocytes at the edge of the scratch also transferred dye much less efficiently at 24 h, in a manner dependent on PKC. However, keratinocyte migration to fill the scratch required early (within <6 h) gap junctional communication. Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.

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Migration of scratch-wounded keratinocytes treated with inhibitors of gap junctional communication or PKC. (A) HFKs were either pretreated for 2 h with 15 μM carbenoxolone (CBX-0h) or 200 nM bisindolylmaleimide (BIM-0h), or untreated (CON), and then were scratch wounded and incubated for 48 h. In some cases, 15 μM CBX or 200 nM BIM was added 6 h after scratch wounding to previously untreated cultures (CBX-6h and BIM-6h, respectively). Error bars represent standard error of the mean. (B) Relative position of untreated (CON), BIM-treated, or CBX-treated cells immediately after scratch wounding (0 time) compared with 48 h later. The lengths of the dashed lines indicating the migration fronts at 48 h were measured and compared with their respective zero time lengths immediately after scratch wounding. Bar, 100 μm.
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fig5: Migration of scratch-wounded keratinocytes treated with inhibitors of gap junctional communication or PKC. (A) HFKs were either pretreated for 2 h with 15 μM carbenoxolone (CBX-0h) or 200 nM bisindolylmaleimide (BIM-0h), or untreated (CON), and then were scratch wounded and incubated for 48 h. In some cases, 15 μM CBX or 200 nM BIM was added 6 h after scratch wounding to previously untreated cultures (CBX-6h and BIM-6h, respectively). Error bars represent standard error of the mean. (B) Relative position of untreated (CON), BIM-treated, or CBX-treated cells immediately after scratch wounding (0 time) compared with 48 h later. The lengths of the dashed lines indicating the migration fronts at 48 h were measured and compared with their respective zero time lengths immediately after scratch wounding. Bar, 100 μm.

Mentions: Because gap junctional communication is known to regulate many different cellular processes, phosphorylation at S368 could regulate multiple wound healing processes, including proliferation, differentiation, and migration. Previous results by others (Noszczyk and Majewski, 2001; Onuma et al., 2001) and staining of our wounds (unpublished data) have shown that proliferation is most extensive 2–7 d after wounding and, thus, that any link with S368 phosphorylation would likely be indirect. We examined migration of keratinocytes up to 48 h after scratch wounding in the presence of BIM or a gap junctional communication inhibitor, carbenoxolone (CBX; Rozental et al., 2001). 6 h after scratch wounding, essentially no migration had occurred under all conditions (Fig. 5 A), consistent with little change in Cx43 expression in the in situ wounds (Fig. 1, c and d). Treatment of keratinocytes with BIM 2 h before scratch wounding (BIM-0h) significantly (P < 0.001) reduced migration at 24 and 48 h, to ∼70% of the control level at the same time points (Fig. 5 A). If BIM was added 6 h after scratch wounding (Fig. 5 A, BIM-6h), little difference was observed in total migration, compared with the control. However, after 48 h, the migration front in BIM-treated cells was noticeably more irregular than in untreated cells, indicating nonuniform migration/outgrowth (Fig. 5 B). We quantified this difference by measuring the length of a line drawn along the front edge of the leading cells after 48 h of migration, relative to the length of a line drawn along the front immediately after the scratch was performed (Fig. 5 B). Cells treated with BIM had average migration front lengths per millimeter of scratch length of 1.19 (BIM-0h) and 1.30 mm (BIM-6h), and both lengths were significantly (Fig. 5 B, P < 0.0001) longer than in the control (1.07) or after CBX treatment (1.06), implying a potential role for PKC regulation in the organization of migration.


Protein kinase C spatially and temporally regulates gap junctional communication during human wound repair via phosphorylation of connexin43 on serine368.

Richards TS, Dunn CA, Carter WG, Usui ML, Olerud JE, Lampe PD - J. Cell Biol. (2004)

Migration of scratch-wounded keratinocytes treated with inhibitors of gap junctional communication or PKC. (A) HFKs were either pretreated for 2 h with 15 μM carbenoxolone (CBX-0h) or 200 nM bisindolylmaleimide (BIM-0h), or untreated (CON), and then were scratch wounded and incubated for 48 h. In some cases, 15 μM CBX or 200 nM BIM was added 6 h after scratch wounding to previously untreated cultures (CBX-6h and BIM-6h, respectively). Error bars represent standard error of the mean. (B) Relative position of untreated (CON), BIM-treated, or CBX-treated cells immediately after scratch wounding (0 time) compared with 48 h later. The lengths of the dashed lines indicating the migration fronts at 48 h were measured and compared with their respective zero time lengths immediately after scratch wounding. Bar, 100 μm.
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Related In: Results  -  Collection

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fig5: Migration of scratch-wounded keratinocytes treated with inhibitors of gap junctional communication or PKC. (A) HFKs were either pretreated for 2 h with 15 μM carbenoxolone (CBX-0h) or 200 nM bisindolylmaleimide (BIM-0h), or untreated (CON), and then were scratch wounded and incubated for 48 h. In some cases, 15 μM CBX or 200 nM BIM was added 6 h after scratch wounding to previously untreated cultures (CBX-6h and BIM-6h, respectively). Error bars represent standard error of the mean. (B) Relative position of untreated (CON), BIM-treated, or CBX-treated cells immediately after scratch wounding (0 time) compared with 48 h later. The lengths of the dashed lines indicating the migration fronts at 48 h were measured and compared with their respective zero time lengths immediately after scratch wounding. Bar, 100 μm.
Mentions: Because gap junctional communication is known to regulate many different cellular processes, phosphorylation at S368 could regulate multiple wound healing processes, including proliferation, differentiation, and migration. Previous results by others (Noszczyk and Majewski, 2001; Onuma et al., 2001) and staining of our wounds (unpublished data) have shown that proliferation is most extensive 2–7 d after wounding and, thus, that any link with S368 phosphorylation would likely be indirect. We examined migration of keratinocytes up to 48 h after scratch wounding in the presence of BIM or a gap junctional communication inhibitor, carbenoxolone (CBX; Rozental et al., 2001). 6 h after scratch wounding, essentially no migration had occurred under all conditions (Fig. 5 A), consistent with little change in Cx43 expression in the in situ wounds (Fig. 1, c and d). Treatment of keratinocytes with BIM 2 h before scratch wounding (BIM-0h) significantly (P < 0.001) reduced migration at 24 and 48 h, to ∼70% of the control level at the same time points (Fig. 5 A). If BIM was added 6 h after scratch wounding (Fig. 5 A, BIM-6h), little difference was observed in total migration, compared with the control. However, after 48 h, the migration front in BIM-treated cells was noticeably more irregular than in untreated cells, indicating nonuniform migration/outgrowth (Fig. 5 B). We quantified this difference by measuring the length of a line drawn along the front edge of the leading cells after 48 h of migration, relative to the length of a line drawn along the front immediately after the scratch was performed (Fig. 5 B). Cells treated with BIM had average migration front lengths per millimeter of scratch length of 1.19 (BIM-0h) and 1.30 mm (BIM-6h), and both lengths were significantly (Fig. 5 B, P < 0.0001) longer than in the control (1.07) or after CBX treatment (1.06), implying a potential role for PKC regulation in the organization of migration.

Bottom Line: Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance.However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it).Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance. Examination of phosphoserine368 (pS368) in normal human skin tissue using a phosphorylation site-specific antibody showed relatively even distribution throughout the epidermal layers. However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it). Scratch wounding of primary human keratinocytes caused a protein kinase C (PKC)-dependent increase in pS368 in cells adjacent to the scratch, with a time course similar to that found in the wounds. Keratinocytes at the edge of the scratch also transferred dye much less efficiently at 24 h, in a manner dependent on PKC. However, keratinocyte migration to fill the scratch required early (within <6 h) gap junctional communication. Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.

Show MeSH
Related in: MedlinePlus