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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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Gap junctional communication near and distant from the scratch wound edge. (A) Phase (top) and Alexa Fluor 488 fluorescence (bottom) views of dye spread after microinjection (injected cell marked with arrows) of scratch edge (left and middle) or distant cells (right). Bar, 25 μm. (B) Average number of cells that received dye after microinjection into untreated (CON) or 200 nM BIM-treated cells at the scratch edge or distant from the scratch (Internal). Error bars represent standard error of the mean. (C) The ratio of the number of recipient cells for BIM-treated cells over control at the scratch edge or distant from the scratch (Internal).
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fig4: Gap junctional communication near and distant from the scratch wound edge. (A) Phase (top) and Alexa Fluor 488 fluorescence (bottom) views of dye spread after microinjection (injected cell marked with arrows) of scratch edge (left and middle) or distant cells (right). Bar, 25 μm. (B) Average number of cells that received dye after microinjection into untreated (CON) or 200 nM BIM-treated cells at the scratch edge or distant from the scratch (Internal). Error bars represent standard error of the mean. (C) The ratio of the number of recipient cells for BIM-treated cells over control at the scratch edge or distant from the scratch (Internal).

Mentions: Gap junctional communication was assayed by microinjection of Alexa Fluor 488 (with a molecular weight of 570.5) in keratinocytes present in 24-h scratch-wounded cultures, to determine whether phosphorylation at S368 affected the ability of HFKs to communicate. Examples of injections distant from and near the scratch edge and of the resulting dye transfer are shown in Fig. 4 A. In untreated cells (Fig. 4 B, CON), communication was limited to ∼4 cells when dye was injected near the scratch, compared with ∼25 cells in unwounded areas. When BIM was added to the scratch-wounded cells, gap junctional communication adjacent to the scratch occurred to ∼10 cells (Fig. 4 B) and was 2.4-fold more extensive (Fig. 4 C) than for the untreated cells injected at the scratch edge. Because cells injected adjacent to the scratch can only transfer away from the wound, the dye transfer levels would be expected to be less than half the level they are in cells that are injected internal to the wound. Thus, the BIM-treated cells adjacent to the scratch transferred dye nearly as efficiently as cells distant from the wound, whereas BIM had little apparent effect on the extent of dye transfer in cells injected far from the wound. Although Fig. 2 shows low levels of phosphorylation of Cx43 at S368 in cells distant from the scratch, apparently these levels are below a threshold level of phosphorylation necessary for detection of a reduction in gap junctional communication by dye transfer. Given the high specificity of BIM, these results indicate that PKC is likely integral to the wound signaling pathway responsible for the temporal reduction of communication of keratinocytes adjacent to human wounds.


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)

Gap junctional communication near and distant from the scratch wound edge. (A) Phase (top) and Alexa Fluor 488 fluorescence (bottom) views of dye spread after microinjection (injected cell marked with arrows) of scratch edge (left and middle) or distant cells (right). Bar, 25 μm. (B) Average number of cells that received dye after microinjection into untreated (CON) or 200 nM BIM-treated cells at the scratch edge or distant from the scratch (Internal). Error bars represent standard error of the mean. (C) The ratio of the number of recipient cells for BIM-treated cells over control at the scratch edge or distant from the scratch (Internal).
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Related In: Results  -  Collection

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fig4: Gap junctional communication near and distant from the scratch wound edge. (A) Phase (top) and Alexa Fluor 488 fluorescence (bottom) views of dye spread after microinjection (injected cell marked with arrows) of scratch edge (left and middle) or distant cells (right). Bar, 25 μm. (B) Average number of cells that received dye after microinjection into untreated (CON) or 200 nM BIM-treated cells at the scratch edge or distant from the scratch (Internal). Error bars represent standard error of the mean. (C) The ratio of the number of recipient cells for BIM-treated cells over control at the scratch edge or distant from the scratch (Internal).
Mentions: Gap junctional communication was assayed by microinjection of Alexa Fluor 488 (with a molecular weight of 570.5) in keratinocytes present in 24-h scratch-wounded cultures, to determine whether phosphorylation at S368 affected the ability of HFKs to communicate. Examples of injections distant from and near the scratch edge and of the resulting dye transfer are shown in Fig. 4 A. In untreated cells (Fig. 4 B, CON), communication was limited to ∼4 cells when dye was injected near the scratch, compared with ∼25 cells in unwounded areas. When BIM was added to the scratch-wounded cells, gap junctional communication adjacent to the scratch occurred to ∼10 cells (Fig. 4 B) and was 2.4-fold more extensive (Fig. 4 C) than for the untreated cells injected at the scratch edge. Because cells injected adjacent to the scratch can only transfer away from the wound, the dye transfer levels would be expected to be less than half the level they are in cells that are injected internal to the wound. Thus, the BIM-treated cells adjacent to the scratch transferred dye nearly as efficiently as cells distant from the wound, whereas BIM had little apparent effect on the extent of dye transfer in cells injected far from the wound. Although Fig. 2 shows low levels of phosphorylation of Cx43 at S368 in cells distant from the scratch, apparently these levels are below a threshold level of phosphorylation necessary for detection of a reduction in gap junctional communication by dye transfer. Given the high specificity of BIM, these results indicate that PKC is likely integral to the wound signaling pathway responsible for the temporal reduction of communication of keratinocytes adjacent to human wounds.

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