PMA promotes in vitro keratinocyte migration independent
of Stat3. (A) Primary cultured keratinocytes were
subjected to in vitro migration assays in the presence
of the indicated growth factors. Every assay was performed after
treating keratinocytes with mitomycin C to prevent their proliferation.
Stat3+/+ keratinocytes showed strong migration in response
to EGF or HGF (Top) as previously described (10) but not
in response to insulin or IGF-I (Top). In contrast,
Stat3−/− keratinocytes did not migrate in response to any
growth factor tested (third row). PMA synergistically stimulated the
migration of Stat3+/+ (second row) and
Stat3−/− (Bottom) keratinocytes in the
presence of insulin or IGF-I. In addition, Stat3−/−
keratinocytes slightly but significantly migrated in response to PMA
plus EGF (P < 0.05) and considerably to PMA plus
HGF (Bottom). Note that treatment with PMA alone did not
stimulate migration of Stat3+/+ or Stat3−/−
keratinocytes (second and third rows). ND, not done. (Bar = 200
μm.) Quantitative evaluation of cell migration (see Materials
and Methods) is shown below each panel. Significantly different
from the control (*, P < 0.05;
**, <0.01) as determined according to Student's
t test. (B) Although PMA plus
insulin-induced migration of wild-type keratinocytes was completely
canceled by GF109203X (5 μM), a specific PKC inhibitor,
Stat3-dependent (EGF- or HGF-induced) migration was insensitive to this
inhibitor, indicating that Stat3 signaling is independent of PKC
activation. Black bars, hatched bars, untreated and GF109203X-treated,
respectively. Significantly different from inhibitor-free control
(**, P < 0.01) according to
Student's t test. NS, not significantly different from
inhibitor-free controls.