Scientific Interests
Our laboratory is interested in the molecular mechanisms that control the self-renewal of epithelial cell stem cell populations versus their irreversible commitment to differentiation. Understanding the balance between epithelial self-renewal and differentiation is of great importance for normal organ morphogenesis, homeostasis and for the development of novel approaches of tissue regeneration and gene therapy. Additionally, transformation of epithelial cells is closely linked to disturbances of their normal growth/differentiation program. An elucidation of this program and its possible alterations are of great potential impact for new therapeutic approaches to human tumors, which are mostly of epithelial origin.

Description of Ongoing Research
Our ongoing research aims to define if the PI3-kinase/Akt pathway plays a major role in the balance between self-renewal and differentiation of keratinocyte stem cells of stratified epithelia, in addition to being a key factor for the execution of the keratinocyte terminal differentiation program (Calautti et al, 2005). To carry out this research, it is essential to isolate and characterize subpopulation of keratinocytes endowed with stem cell properties. Presently, no reliable stem cell marker is available for human keratinocyte stem cells, and therefore their identification is retrospective and based on the ability of these cells to give rise to “holoclones”, stem-derived cell colonies that can be recognized on the basis of mophological and functional parameters. We are currently evaluating how the expansion of these stem cells in vitro can be facilitated by genetic or pharmacological manipulation of the PI3-kinase/Akt pathway, using chemical inhibitors, or via the use of lentivirus expression vectors that overexpress or silence components of this pathway. Our most recent research is aimed to define the role of individual Akt isoforms in keratinocyte stem cell maintenance and/or differentiation. The optimization of grafting procedures to graft human stem cells in immunodeficient mice will permit to analyze the capacity of these cells to generate mature human epithelial tissues in vivo. The next steps of this project will attempt to identify the molecular mechanisms that mediate the downstream function of the PI3-kinase/Akt pathway in keratinocyte stem cells; this objective will be pursued following two complementary approaches: a) identification of Akt substrate in keratinocyte stem cells using phospho-proteomic techniques; b) identification of the functional relationships between the PI3-kinase/Akt pathway with other molecules playing important roles in stem cell regulation, such as the Wnt-beta-catenin pathway, the Notch pathway, the p63 protein and the transcription factors of the Forkhead family.

Results
The PI3-kinase/Akt pathway is active in the stem cell “niches” of stratified epithelia, in vivo. In addition to the basal (deeper) layer of the epidermis, keratinocyte stem cells are located in specific areas of the epithelia, such as the “bulge” of the hair follicle, and the limbus area of the cornear epithelium. Stem cells in these restricted areas are fundamental to assure the physiological cell turnover within mature epithelia, and for tissue regeneration following an external damage. Using antibodies specifically recognizing the active form of Akt, we have previously shown that this signaling molecule is active in the differentiated layers of the epidermis (Calautti et al., 2005). We have also noted that active Akt is present in the “bulge” of the hair follicle, a small group of cells forming a swelling below the insertion of the sebaceous gland on the hair follicle (Fig. 1). Several previous studies have reported that pluripotent stem cells, capable to regenerate the hair follicle, sebaceous glands and epidermis reside in this region.

Figure 1: Active Akt is localized at the bulge of hair follicles. The figure shows an immunohistochenical analysis of the skin and its adnexa (hair follicles and sebaceous glands) stained with antibodies that detect proliferating cells (K14, in green) and active Akt (in red). The arrows indicate the bulge, in which stem cell are found. The yellow/orange signal indicates cells that stain positive to both antibodies.

We have also examined the localizaton of active Akt in the human limbal/corneal epithelium. The cornea is separated from the adjacent conjunctival epithelium by a transition zone named “limbus”. It is well known that the human corneal epithelium is unable to self renew when separated from the limbus, thus the limbus containes the stem cells that are required for the normal cell turnover in the cornea. Immunohistochemical analysis of the limbal-corneal epithelium (Fig. 2) reveals that active Akt is specifically localized in groups of cells in the deeper layers of the limbus epithelium, where stem cells are located. In the cornea, which is devoid of stem cells, no active Akt can be detected.

Figure 2: Active Akt is found in the limbus but is absent in the corneal epithelium. Immunohistochemical analysis of the human limbal-corneal epithelium. Active Akt (in red) is specifically detected in basal cells of the the limbal area (A, B, C). The arrowhead in panel C indicates the transitional zone between the limbus and the cornea. Note that corneal epithelial cells positive to the staining for the differentiation marker keratin-3 (in green) do not show detectable levels of active Akt (see areas on the right of the arrow in panels C and D).

We have been able to isolate keratinocytes endowed with stem cell functions from the human limbus-corneal epithelium and the interfollicolar epidermis. These cell populations display stem cell properties in vitro such as high self-renewal potential and clonogenic ability, as well as the ability to raise progenies of cells able to differentiate into mature epithelial sheets. In particular, keratinocyte stem cells can be identified by their ability to raise “holoclones”, clones of cells with a specific morphology that are considered to be the only type of keratinocyte clones generated by stem cells (Figure 3), which contain stem cells capable of sustaining for the lifetime the high cellular turnover of stratified epithelia, if engrafted into human patients in clinical settings.

Figure 3: Holoclones, stem cell-derived keratinocyte clones. The figures shows the great clonogenic ability of cells derrived from two keratinocyte holoclones enriched of stem cells (H1, H2), compared to the colony-forming capacity of keratinocyte populations devoid of stem cells (M1, M2). Keratinocyte cultures enriched of holoclones are used successfully in cell-based therapies for severe burns and corneal defects.

Notably, keratinocyte cultures derived from holoclones, besides expressing high levels of proteins involved in keratinocyte self-renewal and proliferation potential such as Np63 and Bmi1, also showed high levels of activity of the PI3-kinase/Akt signaling pathway, as compared to keratinocyte cultures derived from clones void of stem cells.

Support
Enzo Calautti is an Assistant Telethon Scientist of the Dulbecco Telethon Institute. This Research Unit is supported by the Telethon grant TCP06001 to Enzo Calautti, and in part, by the “Converging Technologies” grant of the Piemonte Region “Modeling Oncogenic Pathways: from Bioinformatics to Diagnosis and Therapy”.

1. Li J, Baxter RM, Weiner L, Goetinck PF, Calautti E, Brissette JL (2007). Foxn1 promotes keratinocyte differentiation by regulating the activity of protein kinase C. Differentiation 75(8):694-701
   
   
2. Calautti E., Li J., Saoncella S., Brissette J.L. and Paul F. Goetinck (2005) Phosphoinositide 3-kinase signaling to Akt promotes keratinocyte differentiation versus death The Journal of Biological Chemistry 280, 32856-32865
   
   
3. Calautti E., Grossi M., Aoyama Y., Li, J., Pirro M., Mammucari C., and G. Paolo Dotto (2002) Fyn tyrosine kinase is a downstream mediator of Rho/PRK2 function in keratinocyte cell-cell adhesion The Journal of Cell Biology 156 (1), 137-148
   
   
4. Calautti E., Cabodi S., Stein P.L., Hatzfeld M., Kedersha N.and G.P. Dotto (1998) Tyrosine phosphorylation and Src-family kinases control keratinocyte cell-cell adhesion The Journal of Cell Biology 141(6), 1449-1465
   
   
5. Di Cunto F., Calautti E., Topley G., Hsiao J., Ong L. and G.P. Dotto (1998) Citron Rho-Interacting Kinase, a novel tissue-specific ser/thr kinase encompassing the Rho-Rac-binding protein citron The Journal of Biological Chemistry 273, 29706-29711
   
   
6. Calautti, E., Missero, C, Stein, P. L., Ezzel, R. M. and G.P. Dotto (1995) Fyn tyrosine kinase is involved in keratinocyte differentiation control Genes & Development 9, 2279-2291