Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C

Abstract

Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the alpha, delta, eta, and zeta isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the alpha, delta, and eta isoforms. Overexpression of the eta isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The eta-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the eta-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the eta isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative eta isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1alpha,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the delta isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the alpha and zeta isoforms did not. These findings indicate that the eta and delta isoforms of PKC are involved crucially in squamous cell differentiation.

FIG. 1

Expression of PKC isoforms in adenovirus-infected normal human keratinocytes. (A) Immunofluorescence staining of the η isoform in the Ax-PKCη-infected cells. Normal human keratinocytes were infected with Ax-lacZ (lacZ) or Ax-PKCη (η) for 48 h (MOI = 6). (B) Time course of the immunoblots of the η isoform protein in Ax-PKCη-infected cells (MOI = 6). (C) Immunoblotting of PKC isoforms in adenovirus-infected cells. Cells were infected with Ax-lacZ (lacZ), Ax-PKCη (η), Ax-D/NPKCη (D/Nη), Ax-PKCα (α), Ax-PKCδ (δ), and Ax-PKCζ (ζ) for 48 h (MOI = 6).

FIG. 2

Kinase activity in adenovirus-infected cells. (A) Autophosphorylation of the η isoform expressed in COS1 cells. COS1 cells were infected with Ax-lacZ (lacZ; MOI = 6), Ax-PKCη (η; lane 2, MOI = 3; lane 3, MOI = 6), or the dominant-negative mutant of PKCη (D/Nη; MOI = 6) for 48 h. The extracts were immunoprecipitated with antibody against the η isoform and reacted with [γ-³²P]ATP; this was followed by SDS-PAGE and autoradiography. The arrowhead indicates the position of the η isoform. Molecular weight (MW) standards in thousands (K) are shown on the left. (B) Kinase activities of the cells overexpressing the α, δ, η, and ζ isoforms. Partially purified cell extracts were incubated for kinase assay in the presence of PS (50 μg/ml) plus TPA (50 ng/ml) or CS (25 μM). (C) MOI-dependent kinase activity of the Ax-PKCη-infected normal human keratinocytes. Partially purified cell extracts were incubated for assay of kinase activity in the presence (hatched bars) or absence (stippled bars) of PS (50 μg/ml) plus TPA (50 ng/ml).

FIG. 2

Kinase activity in adenovirus-infected cells. (A) Autophosphorylation of the η isoform expressed in COS1 cells. COS1 cells were infected with Ax-lacZ (lacZ; MOI = 6), Ax-PKCη (η; lane 2, MOI = 3; lane 3, MOI = 6), or the dominant-negative mutant of PKCη (D/Nη; MOI = 6) for 48 h. The extracts were immunoprecipitated with antibody against the η isoform and reacted with [γ-³²P]ATP; this was followed by SDS-PAGE and autoradiography. The arrowhead indicates the position of the η isoform. Molecular weight (MW) standards in thousands (K) are shown on the left. (B) Kinase activities of the cells overexpressing the α, δ, η, and ζ isoforms. Partially purified cell extracts were incubated for kinase assay in the presence of PS (50 μg/ml) plus TPA (50 ng/ml) or CS (25 μM). (C) MOI-dependent kinase activity of the Ax-PKCη-infected normal human keratinocytes. Partially purified cell extracts were incubated for assay of kinase activity in the presence (hatched bars) or absence (stippled bars) of PS (50 μg/ml) plus TPA (50 ng/ml).

FIG. 3

Morphology of normal human keratinocytes overexpressing the η and δ isoforms. Cells were infected with Ax-lacZ (lacZ), Ax-PKCη (η), or Ax-PKCδ (δ) at an MOI of 12 and cultured for 48 h.

FIG. 4

Growth inhibition by the η isoform of normal human keratinocytes but not of normal human fibroblasts. Cells were infected on day 1 and cultured for 5 days. Cell growth was monitored by the MTT assay. Values represent means ± standard deviations of three cultures. The experiments were repeated three times with reproducible results. (A) Normal human keratinocytes. Symbols: ○, no infection; •, Ax-lacZ (MOI = 12); ■, Ax-PKCη (MOI = 3); □, Ax-PKCη (MOI = 6); ┌, Ax-PKCη (MOI = 12). (B) Normal human fibroblasts. Symbols: ○, no infection; •, Ax-lacZ (MOI = 12); ■, Ax-PKCη (MOI = 12).

FIG. 5

Inhibition of DNA synthesis (A) and cell cycle progression (B) by overexpression of the η isoform in normal human keratinocytes. (A) Cells were infected with Ax-lacZ (lacZ) or Ax-PKCη (η) for 44 h and then incubated for 4 h with BrdU. Values are means ± standard deviations of triplicate determinations and are shown as a percentage relative to the value for noninfected cells. (B) Cells were infected with Ax-lacZ (lacZ) or Ax-PKCη (η) at an MOI of 12 for 24 h. Cell cycle distribution was determined by flow cytometry.

FIG. 6

Effect of various PKC isoforms on DNA synthesis in normal human keratinocytes. Cells were infected with Ax-lacZ (lacZ), Ax-PKCη (η), Ax-PKCα (α), Ax-PKCδ (δ), Ax-PKCζ (ζ), or Ax-D/Nη (D/Nη) for 44 h and then incubated for 4 h with BrdU. Values represent means ± standard deviations of triplicate determinations and are shown as a percentage relative to the value for noninfected cells (−). Asterisks indicate a significant difference from value of control cells (−), P < 0.01.

FIG. 7

Expression of TGase 1 in normal human keratinocytes overexpressing the η isoform. (A) Northern blot analysis of TGase 1. Normal human keratinocytes were either noninfected (−) or infected with Ax-lacZ (lacZ) or Ax-PKCη (η) for 36 h (MOI = 12). (B) Immunofluorescence staining of TGase 1 protein. Cells were infected with Ax-lacZ (lacZ) or Ax-PKCη (η) for 60 h (MOI = 12) and stained by indirect immunofluorescence.

FIG. 8

Activation of TGase 1 in normal human keratinocytes overexpressing the η isoform. (A) Time course of the induction of TGase 1 activity. Normal human keratinocytes were infected with Ax-lacZ (□) or Ax-PKCη (■) at an MOI of 12, and cell lysate was extracted at the indicated times. Values are means ± standard deviations of triplicate determinations. (B) MOI-dependent activation of TGase 1 by η and δ isoforms. Cells were infected with Ax-lacZ (lacZ), Ax-PKCη (η), or Ax-PKCδ (δ) at the indicated MOIs for 48 h.

FIG. 9

Effect of various PKC isoforms on TGase 1 activity. (A) Normal human keratinocytes were noninfected (−) or infected with Ax-lacZ (lacZ), Ax-PKCη (η), Ax-PKCα (α), Ax-PKCδ (δ), or Ax-PKCζ (ζ) at an MOI of 12 for 48 h. The results show the means ± standard deviations of three cultures. (B) Effect of TPA on TGase 1 activity in the α or ζ isoform-overexpressed cells. Normal human keratinocytes were infected with Ax-lacZ (lacZ), Ax-PKCα (α), or Ax-PKCζ (ζ) at an MOI of 12 for 48 h. TPA (2 ng/ml) was added at 24 h after infection. The results shows the means ± standard deviations of three cultures. (C) Effect of ectopic expression of the η isoform on TGase 1 activity in COS1 cells. COS1 cells were transfected with the TGase 1 gene with or without the PKCη gene using SRD isoform expression vector. TGase 1 activity was measured 48 h after transfection with or without the treatment with 100 nM TPA.

FIG. 10

Suppression of differentiation by the dominant-negative η isoform. Twelve hours after infection with Ax-lacZ (stippled bars) or Ax-D/Nη (hatched bars) at an MOI of 12, normal human keratinocytes grown in KGM were exposed to CaCl₂ (Ca²⁺; 0.2 mM), 1α,25-dihydroxyvitamin D3 (Vit.D3; 12 nM), or TPA (1 ng/ml) for 24 h; measurement of TGase 1 activity followed. Values shown are the means ± standard deviations of three cultures. Asterisks indicate values that are significantly different from the values of Ax-lacZ-infected cells (∗, P < 0.05; ∗∗, P < 0.01).