(74c) JNK Regulates Rigidity-Dependent Adherence Junction Formation of Epithelia in Vivo and in Vitro

Introduction:
In previous work we showed
JNK phosphorylated b-catenin and regulated adherent junction (AJ) formation in
human primary keratinocytes (hKC) and ME180
epithelial cancer cells.  Inhibition
of JNK activity caused translocation of E-cadherin/b-catenin complex to
cell-cell contact sites leading to formation of AJ. It is well known that there
is cross talk between cell-cell and cell-substrate adhesion as manifested by
inhibition of cadherin function upon integrin engagement. This delicate balance between integrin and
cadherin signaling was found to be regulated by substrate rigidity, which also regulates cell spreading, migration,
proliferation and stem cell differentiation and tissue maintenance. Extracellular matrix stiffening has been
shown to increase integrin expression and drive malignant behavior of tumor
cells through Rho-mediated cytoskeletal tension. Tumor invasiveness was also
associated with reduced E-cadherin expression
and
dissolution of AJ. Here we hypothesized that JNK may regulate cell-cell
adhesion in a manner that depends on substrate mechanics.

Materials and Methods: Isolation and
culture of primary keratinocytes and preparation of skin equivalent were
performed as described previously¹. shRNA Targeting the jnk1, jnk2 and jnk1/2 mRNAs was cloned
downstream of the H1 promoter of pLVTHM lentiviral vector. A fusion protein of JNK and its upstream activator
MKK7 was cloned in p-TRIP-Z vector. Polydimethylsiloxane
(PDMS) and a crosslinked siloxane
polymer were used as 9:1 (w/w, base to crosslinker)
for hard substrates (1MPa) and 9:0.2 for soft substrates (16 kPa).

Results
and Discussion: In vitro 2D studies show that the substrate rigidity
regulates JNK activity of hKC. The levels of p-JNK
were significantly reduced in the hKC on soft
substrates. E-cadherin and b-catenin, two components of AJ complex,
co-localized at the cell-cell contact. However, the expressions of p-FAK and p-Paxillin, two of the focal adhesion (FA) markers, at cell
protrusions were significantly
reduced. hKC with
constitutive active JNK had well developed FA but could not form AJ on the soft
substrate. However, hKC knocked down JNK1, JNK2, or
JNK1/2 formed AJ even on the rigid substrates. Notably, our observations
extended in vivo, where we observed a negative correlation between AJ
formation and JNK activity in human foreskin epidermis and bioengineered skin.
Keratinocytes closer to basal layer (stiff substrate) expressed more p-JNK and
p-cJun and lacked AJ. However, keratinocytes on upper suprabasal layers of the epidermis (soft substrate) formed
tight AJ but expressed low levels of p-JNK and p-cJun
(Fig.1A-B). To test
if the stiffness gradient may in turn determine the gradients of p-JNK and AJ
formation observed across the epidermis, we increased the stiffness of the
dermal matrix using a natural crosslinker genipin. Interestingly, expression of p-JNK and p-cJun extended to the upper suprabasal
layers of the epidermis grown on stiffer dermis. Accordingly, the levels of
E-cadherin and b-catenin at the cell-cell contact sites were significantly
reduced even in the upper suprabasal layers (Fig.1C). To verify
that the disruption of AJ in the suprabasal cells was
due to JNK activation, we generated epidermis with hKC
overexpressing the constitutively active JNK1 (MMK7-JNK1 fusion protein). Immunostaining of
tissue sections for E-cadherin and b-catenin showed that MKK7-JNK1 bioengineered epidermis exhibited
weak and disorganized AJ
(Fig.1D). In
agreement, staining for p-JNK and p-cJun was extended
in the suprabasal layers of MKK7-JNK1 bioengineered
epidermis. On the other hand, in either JNK1 and JNK2 deficient mouse skin or
bioengineered skin, robust AJ were formed throughout the epidermis even in the
basal layer (Fig.1E-F), which is normally devoid of AJ.

Fig.1 Immunofluorescent
staining of E-cadherin in (A) human foreskin, (B) wild type bioengineered
skin (C) bioengineered skin on genipin-treated dermis, (D) MKK7-JNK1 hKC
bioengineered skin, (E) shRNA
JNK1 bioengineered skin, (F) shRNA
JNK2 bioengineered skin. View: x63 scale =10µm

Conclusions:  Our
results clearly suggest that formation of AJ is regulated by the activity of
JNK, which in turn may be regulated by substrate stiffness, ultimately
resulting in molecular gradients that may affect tissue development, wound
healing and cancer progression.   

Reference: 1. Andreadis,
S.T., et al. 2001. FASEB J 15, 898-906