(145b) JNK Regulates Cell-Substrate and Cell-Cell Adhesion On Substrates of Varying Compliance

It is well accepted that substrate rigidity can guide the behavior of
cells including spreading, migration, proliferation and differentiation in
vitro (1, 2) and tissue formation and maintenance in vivo (3). Substrate rigidity also has been shown to regulate
the cross talk between
cell-cell and cell-substrate adhesion (4). Our group demonstrated JNK phorphorylates b-catenin and regulates adherent
junction formation, a-catenin/b-catenin interactions and actin
re-organization in epithelial cells (5, 6). Here we provide strong evidence
supporting the role of JNK as a regulator of cell-cell vs. cell-substrate
adhesion on substrates of varying compliance.     
Our in vitro studies show that the substrate rigidity regulates JNK
activity of human primary keratinocytes. The levels of p-JNK were significantly
reduced in the keratinocytes within 24 hrs after seeded on soft substrates (p-JNK1,
12±7%; p-JNK2, 23±9% of plastic wells). In the cells seeded on soft substrate,
E-cadherin and b-catenin, two components of adherent junction complex, co-localized
at the cell-cell contact, however, the expressions of p-FAK and p-Paxillin, two
of the focal adhesion markers, at cells protrusion were reduced.
Keratinocytes with constitutive active JNK could not form adherent junction on
the soft substrate, and the focal adhesion was still detectable. However, keratinocytes
knocked down JNK1, JNK2, or JNK1/2 form adherent junction even on the rigid plastic
surface. In vivo, the negative correlation (R=-0.89) between adherent
junction formation and JNK activity has been demonstrated in human foreskin and
wild type mouse skin tissue. Keratinocytes closer to basal layer express more
p-JNK and p-c-Jun, however, the adherent junctions did not formed properly. The
focal adhesion markers, p-FAK and p-paxillin, were prominently expressed in the
basal layer keratinocytes of skin epidermis, which is consistent with b1
integrin subunit distribution in human skin. The JNK1^-/- mouse skin epidermis
and siRNA JNK1 bioengineered skin contained fewer layers of keratinocytes (mouse
skin 3 vs.7, skin equivalents 6 vs.12). And compared to wild
type, the epidermis is much thinner (mouse skin 33±2%, p<0.05; skin equivalents
27±2%, p<0.05 of WT). However JNK2^-/- mouse skin and siRNA JNK2 skin
equivalent contain similar layers compared to wild type, and the epidermis is
thicker than WT (mouse skin:116±5%, p>0.05, skin equivalents:130±8%, p<0.05
of WT). In either JNK1 and JNK2 deficient mouse skin or bioengineered skin, adherent
junction is properly formed even in the basal layer confirming our results in
vitro.

In conclusion,
keratinocytes on rigid substrate (in vitro) or close to dermis (in
vivo) express high levels of p-JNK, which inhibits adherent junction
formation. On the contrary, keratinocytes on soft substrates or at suprabasal
cell layers showed low p-JNK level, lack of focal adhesions and enhanced
adherent junction formation. In summary, JNK
plays a role as a switch regulating the cross talk between adherent junction
and focal adhesion both in vitro and in vivo models.

Acknowledgements: 
This work was supported
by grants from NIH (R01 EB000876) and NSF (BES-0354626) to S.T.A. We thank Dr.
Roger J. Davis (UMass Medical School) for kindly providing the JNK1^-/-
and JNK2^-/- mouse skin samples.

References:

1.            Buxboim A, et al. 2010 Nat Methods 7:695-697

2.            Joy
A, et al. 2011 Langmuir 27:1891-1899

3.            Guo
WH, et al. 2006 Biophys J 90:2213-2220

4.            Tsai
J, et al.2009 Biophys J 96:L39-41

5.            Lee
MH, et al. 2009 FASEB J 23:3874-3883

6.            Lee
MH, et al. 2010 FASEB J 25:613-623

7.            Koria
P, et al. 2006 J Invest Dermatol 126:1834-1841