270977 JNK Regulates Rigidity-Dependent Adherence Junction Formation of Epithelia in Vivo and in Vitro

Monday, October 29, 2012: 1:06 PM
Somerset East (Westin )
Hui You, Aishwarya Ranganathan and Stelios T. Andreadis, Department of Chemical and Biological Engineering, University at Buffalo, the State University of New York, Amherst, NY

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


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