Role of protin kinases in thein vitrodifferentiation of human epidermal HaCaT cells

Abstract

Different chemicals that specifically and selectively inhibit or activate protein kinases have been used to define the possible roles of these enzymes in the different steps of epidermal differentiation. Using HaCaT keratinocytes as a model, and under conditions in which cell proliferation is minimally affected, we found that tyrosine kinase inhibition leads to an inhibition of early (spinous; keratin k10 expression) and late (granulosum; involucrin expression) differentiation processes. cGMP- and cAMP-dependent protein kinases appear to modulate the transition from spinous to granular differentiation, a process which seems to be negatively controlled by protein phosphatases. Finally, enzymes belonging to the protein kinase C family appear to facilitate the transition from spinous to granular differentiation programmes while inhibiting the early steps of epidermal differentiation.

Modulation of Cell Proliferation by Cytokeratins K10 and K16

“…To study possible specific functions of keratins, we transfected human HaCaT keratinocytes with plasmids coding for K10 (specific to differentiating cells of orthokeratinized epithelia), K13 (specific for differentiating noncornified stratified epithelia), K14 (characteristic of basal cells of all stratified epithelia), and K16 (induced in epidermal cells by hyperproliferative stimuli). HaCaT cells were initially chosen as recipients for these keratins because they are not transformed, retain many epidermal differentiation characteristics, and have been extensively used in vitro as a human keratinocyte model (2,4,9,21,22). On the other hand, epidermal keratinocytes are particularly interesting for these purposes, as they present at least three differentiation stages associated with the expression of specific keratin pairs: mitotic basal cells synthesize K5-K14, terminally differentiating keratinocytes express the K1-K10 pair, and activated hyperproliferative keratinocytes induce keratins K6 and K16.…”

Inhibition of Protein Kinase B (PKB) and PKCζ Mediates Keratin K10-Induced Cell Cycle Arrest

“…To determine whether K10 also interacts with Akt and PKC in vivo, we analyzed the cellular distribution of these kinases during keratinocyte differentiation. Since a small proportion HaCaT cells spontaneously differentiate and induce K10 expression (4,21,27), we first analyzed the distribution of endogenous Akt and PKC in these spontaneously differentiating cells by double immunofluorescence. It was found that both Akt (Fig.…”

“…HaCaT, PB, and MCA3D keratinocytes and C33A and BMGEϩH cells were cultured as previously described (21)(22)(23)(24)(25). For immunofluorescence analysis after transfection, cells were cultured, fixed, and stained as described previously (22-24) using antibodies against keratin K10 (undiluted supernatants of mouse monoclonal antibody [MAb:] LH1, LH2, or LH3 or 1/40-diluted K8.60 mouse MAb) (21)(22)(23)(24)(25).…”

“…Controls, including incubation with preimmune serum, omitting the primary antibody, and preincubation with the immunizing peptide, were routinely performed. Secondary antibodies specific for multiple-labeling purposes were purchased from Jackson Immunoresearch Labs and used as described previously (21)(22)(23)(24)(25). Bromodeoxyuridine (BrdU) incorporation and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were performed essentially as previously described (23, 24) using a rat MAb against BrdU (23) and a fluorescein isothiocyanate (FITC)-labeled cell death detection kit (Boehringer Mannheim) in parallel with K10 immunofluorescence (1/40 dilution of antibody K8.60).…”

“…HaCaT, PB, and MCA3D keratinocytes and C33A and BMGEϩH cells were cultured as previously described (21)(22)(23)(24)(25). For immunofluorescence analysis after transfection, cells were cultured, fixed, and stained as described previously (22-24) using antibodies against keratin K10 (undiluted supernatants of mouse monoclonal antibody [MAb:] LH1, LH2, or LH3 or 1/40-diluted K8.60 mouse MAb) (21)(22)(23)(24)(25). PKC (Santa Cruz Biotechnology or Boehringer Mannheim; both rabbit polyclonal antibodies used at 1/200 and 1/400 dilutions, respectively), Akt (Upstate Biotechnology or Santa Cruz Biotechnology; both goat polyclonal antibodies used at 1/250 and 1/300 dilutions, respectively), phosphorylated Akt (Upstate Biotechnology or BioLabs; both rabbit polyclonal antibodies used at 1/200 and 1/100 dilutions, respectively), and a mouse MAb against VSV-G tag diluted 1/100 (Boehringer Mannheim).…”