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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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Related in: MedlinePlus

Changes in Cx43 and pS368 levels 24 h after scratch wounding HFKs. Immunofluorescence for total Cx43 (A) and pS368 (B) staining and their colocalization (C). DAPI nuclear staining (blue) is also included in the overlay. Bar, 50 μm.
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fig2: Changes in Cx43 and pS368 levels 24 h after scratch wounding HFKs. Immunofluorescence for total Cx43 (A) and pS368 (B) staining and their colocalization (C). DAPI nuclear staining (blue) is also included in the overlay. Bar, 50 μm.

Mentions: Primary HFKs were used to allow examination of gap junctional communication and signaling pathways associated with the wounding process. To determine whether wounding of HFKs would affect phosphorylation at S368 consistent with in vivo wounds, we scratch wounded HFK monolayers with a pipette tip in a manner consistent with previous studies (e.g., Yamada et al., 2000). We performed immunofluorescence to determine whether changes elicited by scratch wounding paralleled those found in the in vivo wounds; i.e., whether pS368 levels increased near the scratch edge. Fig. 2 shows immunofluorescence of total Cx43 (A) and pS368 (B) in keratinocytes 24 h after scratch wounding. Although a reduction in Cx43 in the differentiating suprabasal cells was observed in wounds (Fig. 1 f), little change in Cx43 levels was observed near the scratch (Fig. 2 A), possibly because the cultured cells, like basal cells, are not differentiated. The ratio of pS368 to Cx43 signal, most clearly demonstrated in the overlay panel (Fig. 2 C), shows that the relative pS368 levels are highest 1–4 cell diameters from the scratch. Note that the signal for pS368 appears to be plasma membrane–associated and continuous throughout the cell–cell interfaces, similar to the basal cell staining in the in situ wounds (Fig. 1 f, inset). The total Cx43 antibody used for this staining has a preference for the Cx43 present in gap junctions (Solan et al., 2003), so the continuous pS368 staining may represent protein that is not yet assembled into gap junctions. This possibility would be consistent with the observation that PKC activation inhibits the formation of new gap junctions (Lampe, 1994). Further away from the scratch, most of the pS368 signal overlays with the total Cx43 signal.


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)

Changes in Cx43 and pS368 levels 24 h after scratch wounding HFKs. Immunofluorescence for total Cx43 (A) and pS368 (B) staining and their colocalization (C). DAPI nuclear staining (blue) is also included in the overlay. Bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172473&req=5

fig2: Changes in Cx43 and pS368 levels 24 h after scratch wounding HFKs. Immunofluorescence for total Cx43 (A) and pS368 (B) staining and their colocalization (C). DAPI nuclear staining (blue) is also included in the overlay. Bar, 50 μm.
Mentions: Primary HFKs were used to allow examination of gap junctional communication and signaling pathways associated with the wounding process. To determine whether wounding of HFKs would affect phosphorylation at S368 consistent with in vivo wounds, we scratch wounded HFK monolayers with a pipette tip in a manner consistent with previous studies (e.g., Yamada et al., 2000). We performed immunofluorescence to determine whether changes elicited by scratch wounding paralleled those found in the in vivo wounds; i.e., whether pS368 levels increased near the scratch edge. Fig. 2 shows immunofluorescence of total Cx43 (A) and pS368 (B) in keratinocytes 24 h after scratch wounding. Although a reduction in Cx43 in the differentiating suprabasal cells was observed in wounds (Fig. 1 f), little change in Cx43 levels was observed near the scratch (Fig. 2 A), possibly because the cultured cells, like basal cells, are not differentiated. The ratio of pS368 to Cx43 signal, most clearly demonstrated in the overlay panel (Fig. 2 C), shows that the relative pS368 levels are highest 1–4 cell diameters from the scratch. Note that the signal for pS368 appears to be plasma membrane–associated and continuous throughout the cell–cell interfaces, similar to the basal cell staining in the in situ wounds (Fig. 1 f, inset). The total Cx43 antibody used for this staining has a preference for the Cx43 present in gap junctions (Solan et al., 2003), so the continuous pS368 staining may represent protein that is not yet assembled into gap junctions. This possibility would be consistent with the observation that PKC activation inhibits the formation of new gap junctions (Lampe, 1994). Further away from the scratch, most of the pS368 signal overlays with the total Cx43 signal.

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