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Integrin Linked Kinase Production Prevents Anoikis in Human Mesenchymal Stem Cells

Danielle S.W. Benoit1, Margaret C. Tripodi1, James O. Blanchette2, Stephen J. Langer3, Leslie A. Leinwand3, and Kristi S. Anseth4. (1) Department of Chemical and Biological Engineering, University of Colorado, ECCH 111 Campus Box 424, Boulder, CO 80309, (2) Howard Hughes Medical Institute, 424 UCB, Boulder, CO 80309-0424, (3) Department of Molecular, Cellular, and Developmental Biology, University of Colorado, MCDB A418, Campus Box 347, Boulder, CO 80309, (4) Department of Chemical and Biological Engineering, University of Colorado, Howard Hughes Medical Institute, ECCH 128, Campus Box 424, Boulder, CO 80309-0424

The nature of cell adhesion and subsequent signaling via receptor-ligand interactions provides the cell with vital information about its extracellular environment, regulating a variety of events, including tissue evolution, cell migration, and even survival [1,2]. For instance, anchorage-dependent osteoprogenitor cells, such as mesenchymal stem cells (MSCs), require integrin interactions in order to survive [3]. Indeed, the interaction of epithelial and endothelial cells with the extracellular matrix (ECM) inhibits default apoptotic pathways, which become activated if cell-ECM interactions are disrupted [4], and this process is termed anoikis. Thus, cell-ECM interactions are paramount for maintaining cell viability and a critical aspect of designing niches for three-dimensional cell culture and tissue regeneration.

The link between integrins and the ECM takes place at focal adhesions. Many accessory cytoskeletal proteins concentrate at focal adhesions, as do various signaling proteins, some of which are primarily related to cell adhesion. Activated protein kinase B (PKB/Akt) plays a critical role in regulating adhesion-mediated cell survival signals [5,6]. Integrin linked kinase (ILK), first identified as an integrin β1-cytoplasmic domain binding protein [7], enhances phosphorylation of PKB/Akt, which is essential for PKB/Akt activation [8] either directly or through a complex involving PINCH-1 [8]. Therefore, ILK functions as a pivotal effector in the transduction of signals from the ECM, regulating, among others, the apoptotic pathway.

Numerous groups are exploring strategies to design synthetic extracellular matrix analogs that present cells with integrin-specific ligands, such as the ubiquitous Arg-Gly-Asp (RGD) peptide sequences, to promote cell-materials interactions. In this respect, poly(ethylene glycol) (PEG) hydrogels are often used as the base material, as non-specific protein adsorption to PEG is minimal. Thus, the effect of targeted ligands on cell interactions can be selectively studied. Not surprisingly, in the absence of adhesive ligands, most anchorage-dependent cells undergo anoikis. For example, a dramatic decrease from 99% to 20% in the survival of mesenchymal stem cells encapsulated in pure PEG gels is observed over the course of two weeks [9].

Here, we are interested in better understanding the intracellular processes that lead to anoikis and then develop strategies that enable the study of cell functions in the absence of extracellular interactions. Specifically, human MSCs (hMSCs) were photoencapsulated in RGD-presenting PEGDA hydrogels and ILK production was monitored. Furthermore, to determine whether ILK production is sufficient for survival, hMSCs were infected with an adenovirus expressing ILK. ILK expression was first optimized and ensured to be long-term and ILK activity was monitored through PKB/Akt phosphorylation. hMSCs were infected with both ILK-expressing and cyclization recombinase (CRE)-expressing (as a negative control) viruses. These cells and uninfected cells were encapsulated in PEGDA hydrogels. In addition, as a positive control, uninfected cells were encapsulated in RGD-modified PEGDA hydrogels. Encapsulated cells were cultured for up to 7 weeks and assessed for viability at days 2, 5, 14, 28, and 49. Results show that the viability is greater in cells infected with ILK-expressing virus than for CRE-infected cells or for uninfected cells encapsulated in PEGDA hydrogels, especially as the study progresses past 5 days. In addition, RGD-modified gels supported viability of hMSCs, as previously reported. RGD-modified gels and ILK-infected hMSCs in ‘blank slate' gels exhibited 80% viability after 7 weeks of culture. Conversely, uninfected cells or CRE-infected cells encapsulated in PEGDA suffered significant cell death, where only about 40% of the cells remained viable at 7 weeks.

The samples from the LIVE/DEAD assay were homogenized and immunoblots were utilized to confirm that our cells are producing ILK and the downstream product, pAKT is also affected. ILK production by ILK-infected cells encapsulated in PEGDA and activity, as monitored by pAKT, was at a similar level to uninfected cells encapsulated in RGD-modified PEGDA over the entire study. In addition, control cells (CRE-infected cells and uninfected cells encapsulated in PEGDA) showed production and activity of ILK at levels about 20-fold and 15-fold less, respectively, than the production by ILK-infected or RGD-modified PEGDA encapsulated cells.

The trend of viability as well as ILK production and activity for cells infected with ILK-producing virus is statistically the same to that of uninfected cells encapsulated in RGD-modified PEGDA, which confirms the hypothesis that infecting hMSCs with ILK-expressing virus rescues the cells over time in a manner similar to RGD. Thus, ILK production in infected hMSCs essentially mimics ILK activity implicated in RGD-mediated cell survival, leading to the suppression of anoikis. Additionally, the viability of hMSCs infected with CRE-expressing virus decreases at statistically the same rate as uninfected hMSCs, indicating that infection of CRE does not interfere with the anoikis pathway.

1. Koenig, A. and D. Grainger. 2002. In: A. Dillow, A. Lowman. Biomimetic Materials and Design. Marcel Dekker, Inc., New York, p. 187.

2. Longhurst, C. and L. Jennings. 1998. Integrin-mediated signal transduction. Cellular and Molecular Life Sciences, 54: 514-526.

3. Ishaug, S.L., R.C. Thomson, A.G. Mikos, and R. Langer. 1995. Biomaterials for organ regeneration. In: R.A. Meyers, R.A. Meyers. Molecular biology and biotechnology: a comprehensive desk reference. New York, p. 86-93.

4. Howe, A, A.E. Aplin, S.K. Alahari, and R.L. Juliano. 1998. Integrin signaling and cell growth control. Current Opinion in Cell Biology, 10: 220-231.

5. Alahari, S.K., P.J. Reddig, and R.L. Juliano. 2002. Biological aspects of signal transduction by cell adhesion receptors. International Reviews in Cytology, 220: 145-184.

6. Nicholson, K.M. and N.G. Anderson. 2002. The protein kinase B/Akt signaling pathway in human malignancy. Cellular Signaling, 14: 381-395.

7. Hannigan, G.E., C. Leung-Hagesteijn, L. Fitz-Gibbon, M.G. Coppolino, G. Radeva, J. Filmus, J.C. Bell, and S. Dedhar. 1996. Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase. Nature, 379: 91-96.

8. Wu, C. 2006. PINCH, N(i)ck and the ILK: network wiring at cell-matrix adhesions. Trends in cell biology, In press.

9. Nuttelman, C.R., M.C. Tripodi, and K.S. Anseth. 2005. Synthetic hydrogel niches that promote hMSC viability. 24: 208-218.