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First published online 27 July 2004
doi: 10.1242/jcs.01277

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3ß1 integrin promotes keratinocyte cell survival through activation of a MEK/ERK signaling pathway

Asha Manohar^*, Swati Ghosh Shome^*, John Lamar, Lee Stirling, Vandana Iyer, Kevin Pumiglia and C. Michael DiPersio

Center for Cell Biology and Cancer Research, Albany Medical College, MC-165, 47 New Scotland Avenue, Albany, New York, NY 12208, USA

View larger version (67K): [in a new window]   Fig. 3. 6ß4-mediated adhesion is not required for keratinocyte survival on LN-5 ECM in the presence of 3ß1. 3-null MK-/- cells (A,B), wild-type MK+/+ cells (C,D), or 3-transfected MK-/- cells (E,F) were seeded onto LN-5 ECM in serum-free medium in the presence of 5 µg/ml of anti-6 blocking antibody GoH3 (B,D,F) or rat IgG2a isotype control antibody (A,C,E). Adherent cells were photographed approximately two hours after plating. Note that MK-/- cells fail to spread due to the lack of 3ß1 (A), and GoH3 completely eliminated MK-/- cell adhesion (B). (G) 24 hours after adhesion, the cells shown in panels A and C-F were lysed and assayed for caspase-3 activation by western blot as described in Fig. 2 (upper panels); cell lines and antibody treatments are indicated above the lanes. Filters were stripped and re-probed for keratin 14 (lower panels, K14). Results are representative of two independent experiments, one of which was performed in duplicate.

View larger version (46K): [in a new window]   Fig. 1. Absence of 3ß1 from keratinocytes leads to increased apoptosis upon serum deprivation. Wild-type MK+/+ cells (A,B), 3-null MK-/- cells (C,D), or 3-transfected MK-/- cells (E,F) were cultured on LN-5 ECM in serum-free medium for two days. Apoptotic cells were identified by TUNEL staining (A,C,E); corresponding phase images are shown (B,D,F, respectively). Examples of apoptotic cells are indicated with arrows in C and D. (G) Flow cytometry was performed and the percent of TUNEL-positive cells was determined for MK-/- cells (MK-/-), or 3-transfected MK-/- cells (MK3) grown in the presence (white bars) or absence (black bars) of serum.

View larger version (48K): [in a new window]   Fig. 2. Absence of 3ß1 from keratinocytes leads to increased caspase-3 activation upon serum deprivation. (A) Wild-type MK+/+ cells (+/+ lanes), 3-null MK-/- cells (-/- lanes), or 3-transfected MK-/- cells (3 lanes) were cultured on LN-5 ECM for 24 hours in the presence of serum (+serum) or in the absence of serum (serum-free). Cell lysates were assayed for caspase-3 activation by western blot. (B) Cells were kept in suspension for 24 hours in the absence of serum (serum-free, susp.), then assayed for caspase-3 activation as in A. Migratory positions of pro-caspase-3 and cleaved forms of caspase-3 are indicated. Results are representative of more than four independent experiments.

View larger version (41K): [in a new window]   Fig. 4. 3ß1 mediates adhesion-dependent activation of FAK/CAS signaling. Wild-type MK+/+ cells (+/+ lanes), 3-null MK-/- cells (-/- lanes), or 3-transfected MK-/- cells (3 lanes) were kept in suspension (suspended) or adhered to LN-5 ECM under serum-free conditions for the times indicated. (A,B) To assay levels of activated FAK, cell lysates were immunoblotted with an antibody against phospho-FAK (Tyr397) (pFAK, upper panel). Blots were stripped and reprobed for total FAK protein (FAK, lower panel). (C) To assay levels of phosphorylated CAS, cell lysates were immunoprecipitated with anti-p130CAS monoclonal antibody, followed by immunoblotting with monoclonal antibody 4G10 against phospho-Tyr (upper panel). Blots were stripped and reprobed for total CAS protein (lower panel).

View larger version (92K): [in a new window]   Fig. 5. Exogenous FRNK expression induces keratinocyte apoptosis on LN-5 ECM. (A,B) Wild-type MK+/+ cells were left uninfected (uninfect. lanes) or infected with adenovirus expressing either GFP-FRNK fusion protein (GFP-FRNK lanes) or GFP only as a control (GFP lanes). Cells were then sub-cultured on LN-5 ECM for 24 hours under serum-free conditions. (A) Cell lysates were immunoblotted with anti-GFP antibody to confirm expression of exogenous proteins; the migratory positions of GFP (27-29 kDa) and GFP-FRNK (68 kDa) are indicated. (B) Cells were assayed for activation of caspase-3 by immunoblotting as described in Fig. 2; migratory positions of pro-caspase-3 and cleaved caspase-3 are indicated. A lower proportion of intact protein was recovered from GFP-FRNK expressing cells due to extensive apoptosis (see panels Cc,d); therefore, a longer exposure of the GFP-FRNK lane is included to emphasize the proportion of caspase-3 that is cleaved. Filters were stripped and reprobed for keratin 14 (K14). (C) Fluorescence of representative fields of cells infected with either GFP (a) or GFP-FRNK (c). Corresponding phase images are shown in b and d, respectively. Arrowheads point to examples of infected (i.e. fluorescent) cells. Note that most cells expressing GFP remained well spread with no signs of apoptosis (a,b), while the vast majority of GFP-FRNK expressing cells displayed an apoptotic phenotype (c,d). Arrows in panels c and d point to uninfected cells, which do not show signs of apoptosis and serve as an internal control.

View larger version (46K): [in a new window]   Fig. 6. 3ß1 is required for adhesion-dependent activation of ERK. (A) Wild-type MK+/+ cells (+/+ lanes), 3-null MK-/- cells (-/- lanes), or 3-transfected MK-/- cells (3 lanes) were kept in suspension for 30 minutes or adhered to LN-5 ECM under serum-free conditions for 15 minutes or 24 hours, as indicated. To assay ERK phosphorylation, cell lysates were immunoblotted with antibodies against phosphorylated ERK (pERK, upper panel) or total ERK (ERK, lower panel). (B) To assay ERK activity, lysates were prepared from suspended cells or from cells adhered to LN-5 for 15 minutes, then tested for in vitro phosphorylation of an Elk-1 substrate (pELK), as described in the methods. (C) MK+/+ cells were adhered to LN-5 ECM and cultured in serum-free medium for 24 hours in the absence of antibody (no Ab), or in the presence of either anti-6 blocking antibody (GoH3) or rat IgG2a isotype control antibody (IgG), as described in Fig. 3. Cell lysates were immunoblotted with antibodies against phosphorylated ERK or total ERK, as described in A.

View larger version (39K): [in a new window]   Fig. 7. MEK/ERK signaling is required for keratinocyte survival on LN-5 ECM. (A) Wild-type MK+/+ cells or 3-null MK-/- cells were cultured on LN-5 ECM in serum-free medium in the presence or absence of the MEK inhibitor U0126 (10 µM), as indicated. Cell lysates were assayed for phosphorylated ERK (pERK, upper panel) or total ERK (ERK, lower panel), as described in Fig. 6. Identical results were obtained in cells cultured for 24 or 48 hours. (B) Wild-type MK+/+ cells or 3-null MK-/- cells were cultured on LN-5 ECM in serum-free medium for 48 hours, in the presence or absence of 10 µM U0126, as indicated. Cell lysates were assayed for caspase-3 activation by western blot, as described in Fig. 2. Results are representative of two independent experiments, one of which was performed in duplicate.

View larger version (47K): [in a new window]   Fig. 8. Expression of activated Ras-V12 promotes MEK/ERK-independent survival of 3-null keratinocytes. 3-null MK-/- cells were infected with adenovirus expressing HA-tagged Ras-V12 (Ras-V12 lanes) or with a control adenovirus expressing ß-galactosidase (ß gal lanes) then sub-cultured on LN-5 ECM for 24 hours in serum-free medium in the presence or absence of 10 µM U0126, as indicated. As controls, uninfected MK-/- cells were cultured in the presence of serum (uninfect., +serum) or in serum-free medium (uninfect., serum-free). (A) To confirm expression of Ras-V12, cell lysates were immunoblotted with anti-HA tag (HA-Ras-V12). ERK activation in Ras V12-infected cells was assayed by probing parallel blots with anti-phospho-ERK (pERK) or total ERK (ERK), as described in Fig. 6. (B) Cells were assayed for activation of caspase-3 by western blot, as described in Fig. 2; migratory positions of procaspase-3 and cleaved caspase-3 are indicated. Filters were stripped and re-probed for keratin 14 (K14). Results are representative of five independent experiments for adenoviral infections; inhibitor treatments were included in two experiments.

View larger version (57K): [in a new window]   Fig. 9. A model for 3ß1-mediated keratinocyte survival at the cutaneous wound edge. (A) In quiescent epidermis, 6ß4 (black integrin) is polarized to the basal cell surface in hemidesmosomes, where it mediates stable adhesion of keratinocytes to LN-5 in the basement membrane. 6ß4 also contributes to maintenance of keratinocyte survival, along with other ECM receptors (see text for discussion); 3ß1 may contribute to survival, as well. (B) During epidermal wound healing, activated keratinocytes at the wound edge disassemble hemidesmomes, and 3ß1 (grey integrin) redistributes to the basal surface where it binds to newly deposited LN-5. In addition to promoting cell migration (Nguyen et al., 2000), 3ß1-LN-5 interactions at the wound edge may contribute to keratinocyte survival following the loss of 6ß4-mediated adhesion.