The Effect of N-Acetyl Cysteine on Myofibroblast Phenotype Expression by Immortalized Keratinocytes, Ker-CT-RasJessica M. Webb1, Pritika Khadka2, and Melville B. Vaughan, Ph. D2

University of Central OklahomaDepartment of Engineering Physics1, Department of Biology2

.

AcknowledgementsThis project was made possible by the OK-LSAMP

and RCSA grants and the CURE-S-STEM scholarship.

Abstract

Recent research activity has focused on the tumor stroma. Tumor stroma are connective tissues containing fibroblasts and myofibroblasts, cells required for wound healing. There is evidence that myofibroblast presence in tumor stroma leads to poor prognosis. Mechanical tension enhances differentiation of myofibroblasts. Myofibroblasts are distinguished from fibroblasts by the assembly of α-sm actin filaments. Transforming growth factor-beta (TGF-β) is the best-known inducer of α-sm actin and is correlated with increased contractility.Precancerous keratinocytes lead to two types of carcinomas. In vitro carcinomas can form through a pathway which involves the up-regulation of the H-ras protein. Ker-CT-Ras demonstrate an epithelial to mesenchymal transition (EMT), meaning the cells begin to demonstrate fibroblast form and function. Fibroblasts generate tension in the dermis during wound healing. So far, Ker-CT-Ras has shown an ability to generate tension in a dermal equivalent with an increased effect in the presence of TGF-β as well as a reduction of this ability when dosed with N-acetyl cysteine (NAC).Grinnell’s stress-relaxation collagen matrix model provided the necessary microenvironment for myofibroblast formation. The model was originally used to investigate properties of fibroblasts, but we have appropriately extended its use to Ker-CT-Ras. Previously, we gathered comparative data on the Ker-CT-Ras matrices to the tension-generating ability of fibroblast matrices (DP-147-H-Tert). Now, we will present preliminary Western blot (W.B.) data examining the production of α-sm actin in Ker-CT-Ras collagen matrices. 

Introduction and Methods

Ker-CT Ker-CT-Ras

Epithelial to mesenchymal transition (EMT) (Vaughan et. al., PLoS ONE 2009)

Grinnell, F. The Journal of Cell Biology, Volume 124,1994

The stress-relaxed collagen matrix (above right) allows in vitro contraction of fibroblast in a synthetic wounded dermis, due to the characteristic fibroblast contraction that is associated with wound healing. Invasive Ker-CT-Ras demonstrate the ability to invade the dermal compartment (above, center) and similarly contract a collagen matrix void of fibroblasts. Staining techniques were used to detect proliferation and alpha smooth muscle actin (α-sm-actin) stress fibers, which are characteristic of myofibroblasts. Coverslips were stained to achieve better image quality than with the released lattice sections. Lattice contraction was measured for best-fit area with ImageJ. The data gave us an understanding of the initial W.B. data. TGF-β was added to the media surrounding the experimental coverslips and lattices prior to staining and during lattice tension generation. W.B. samples were prepared and frozen by Jessica on days 3 and 4 with unreleased lattices. W.B. samples were run by Pritika in a 2-day process.

Literature Cited

1. Grinnell, Frederick. (1994). “Fibroblasts, myofibroblasts, and wound contraction” Journal of Cell Biology. 124: 401-404.

2. Tomasek J.J. et. al. (1992). “Fibroblast contraction Occurs on Release of Tension in attached collagen lattices: Dependency on an Organized Actin Cytoskeleton and Serum”. The Anatomical Record. 232: 359-368.

3. Vaughan, Melville et. al. (2000). “Transforming Growth factor-β promotes the morphological and functional differentiation of the myofibroblast”. Experimental Cell Research. 257: 180-189.

4. Valencia, A. et. al. (2010). “The Significance of Transforming Growth Factor-Beta (TGF-β) on Ker-CT-Ras Using Immortalized Keratinocytes in Cultured Skin Equivalents.”

5. Xu, J., Samy L., and Derynck, R. (2009). “TGF-β-Induced epithelial to mesenchymal transition.” Cell Research. 19: 156-172.

6. Vaughan, M.B. et. al. (2009). “H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents. PLoS ONE 4(11): e7908, doi: 10.1371/journal.pone.0007908

7. Kopp J, Seyham H, et.al. N-Acetyl-L-Cysteine abrogates fibrogenic properties of fibroblasts isolated form Dupuytren’s disease by blunting TGF-β signaling. J. Cell Mol. Med. 2006; 10: 157-165.

*A special thanks to Tobi Odejimi for providing aCT4-H-Tert myofibroblast picture.

Conclusion

Our study shows that Ker-CT-Ras contracts in a collagen lattice and assembles α-sm-actin stress fibers. These previously shown data demonstrate how Ker-CT-Ras has become myofibroblast-like through its tension-generating phenotype. This phenotype has been shown as “blunted” by the presence of TGF-β blocker, NAC. Furthermore, our Western blot analysis gave more evidence for Ker-CT-Ras EMT by indicating α-sm-actin expression in contractile lattices with or without the experimental treatment (TGF-β).

Discussion (continued)

Ker-CT-Ras here do not respond as previously shown. The contraction data indicates that the concentration of TGF-β is too low. The fact that the W.B. shows α-sm-actin at all is intereting because normal keratinocytes do not express α-sm-actin. With more data, these results could give more indication to the EMT of Ker-CT-Ras.

In order to make our results more robust, we will make a concentration curve to find the potency of TGF- β in the stock solution relative to the TGF-β previously used.

Results

Coverslips: Differentiated Myofibroblast(s) from fibroblast* (20x, left) and co-culture (DP147-h-TERT and Ker-CT-Ras) (60x, right);

Solitary Ker-CT-Ras with Tgf-β

and NAC

Ker-CT-Ras, tgf-β and NAC, 60x

30ug of protein was probed for α-sm-actin. The target appeared in Ker-CT-Ras treated with and without TGF-β. Control on left; TGF-β treated on right.

TGF- β is known for up-regulating the expression of α-sm-actin stress fibers, which are produced by fibroblasts. Because of this up-regulation, the fibroblasts release more EDA-fibronectin, which acts as a surfactant and makes the lattices and coverslips “stickier”, an effect that increases the resultant tension. That tension in turn up-regulates general stress fiber production. Ker-CT-Ras, like fibroblasts, have been shown to generate more tension when treated with TGF-β. Contrarily, the W.B. data shows that α-sm-actin is present in relatively the same quantity for day 3 and day 4 lattices, with or without the presence of TGF-β.

Discussion