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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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Immunohistochemistry staining of normal and wounded human skin collected 6, 24, and 72 h after wounding. Serial sections of skin tissue collected at 6, 24, and 72 h after wounding were reacted with antibodies to either Cx43 or pS368, and their localization is indicated by brown DAB staining. The rabbit primary antibody was omitted from the staining procedure for a 24-h wound section as a control (i). Dashed line indicates the basement membrane border between the dermis (lower) and epidermis (upper). Dead cornified cells (uppermost layers) lack Cx43 staining. (c–i) Wound edge is indicated by an arrowhead. Bars, 50 μm.
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fig1: Immunohistochemistry staining of normal and wounded human skin collected 6, 24, and 72 h after wounding. Serial sections of skin tissue collected at 6, 24, and 72 h after wounding were reacted with antibodies to either Cx43 or pS368, and their localization is indicated by brown DAB staining. The rabbit primary antibody was omitted from the staining procedure for a 24-h wound section as a control (i). Dashed line indicates the basement membrane border between the dermis (lower) and epidermis (upper). Dead cornified cells (uppermost layers) lack Cx43 staining. (c–i) Wound edge is indicated by an arrowhead. Bars, 50 μm.

Mentions: Expression of connexin genes is tissue and species specific. In mouse epidermis, at least nine different connexins can be expressed at different levels throughout the skin layers during development (for review see Richard, 2000). Although many of the same proteins are present, the connexin expression pattern in human epidermis is different. The most abundant connexin expressed in human epidermis and cultures of human keratinocytes is clearly Cx43 (Fitzgerald et al., 1994; Di et al., 2001). In human skin, Cx43 is expressed at lower levels in the proliferative basal cell region and at higher levels upon differentiation in the suprabasal layers (Salomon et al., 1994; Lampe et al., 1998). Immunohistochemistry and differential interference contrast microscopy (Fig. 1 a) confirmed extensive Cx43 levels in the suprabasal cells. Basal cells, attached directly to the basement membrane (Fig. 1 a, dashed line), show only low levels of staining for Cx43.


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)

Immunohistochemistry staining of normal and wounded human skin collected 6, 24, and 72 h after wounding. Serial sections of skin tissue collected at 6, 24, and 72 h after wounding were reacted with antibodies to either Cx43 or pS368, and their localization is indicated by brown DAB staining. The rabbit primary antibody was omitted from the staining procedure for a 24-h wound section as a control (i). Dashed line indicates the basement membrane border between the dermis (lower) and epidermis (upper). Dead cornified cells (uppermost layers) lack Cx43 staining. (c–i) Wound edge is indicated by an arrowhead. Bars, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Immunohistochemistry staining of normal and wounded human skin collected 6, 24, and 72 h after wounding. Serial sections of skin tissue collected at 6, 24, and 72 h after wounding were reacted with antibodies to either Cx43 or pS368, and their localization is indicated by brown DAB staining. The rabbit primary antibody was omitted from the staining procedure for a 24-h wound section as a control (i). Dashed line indicates the basement membrane border between the dermis (lower) and epidermis (upper). Dead cornified cells (uppermost layers) lack Cx43 staining. (c–i) Wound edge is indicated by an arrowhead. Bars, 50 μm.
Mentions: Expression of connexin genes is tissue and species specific. In mouse epidermis, at least nine different connexins can be expressed at different levels throughout the skin layers during development (for review see Richard, 2000). Although many of the same proteins are present, the connexin expression pattern in human epidermis is different. The most abundant connexin expressed in human epidermis and cultures of human keratinocytes is clearly Cx43 (Fitzgerald et al., 1994; Di et al., 2001). In human skin, Cx43 is expressed at lower levels in the proliferative basal cell region and at higher levels upon differentiation in the suprabasal layers (Salomon et al., 1994; Lampe et al., 1998). Immunohistochemistry and differential interference contrast microscopy (Fig. 1 a) confirmed extensive Cx43 levels in the suprabasal cells. Basal cells, attached directly to the basement membrane (Fig. 1 a, dashed line), show only low levels of staining for Cx43.

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