Enzo Calautti

Phosphoinositide 3-Kinase Signaling to Akt Promotes Keratinocyte Differentiation Versus Death

Signaling pathways regulating the differentiation program of epidermal cells overlap widely with those activated during apoptosis. How differentiating cells remain protected from premature death, however, is still poorly defined. We show here that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated at early stages of mouse keratinocyte differentiation both in culture and in the intact epidermis in vivo. Expression of active Akt in keratinocytes promotes growth arrest and differentiation, whereas pharmacological blockade of PI3K inhibits the expression of “late” differentiation markers and leads to death of cells that would otherwise differentiate. Mechanistically, the activation of the PI3K/Akt pathway in keratinocyte differentiation depends on the activity of the epidermal growth factor receptor and Src families of tyrosine kinases and the engagement of E-cadherin-mediated adhesion. During this process, PI3K associates increasingly with cadherin-catenin protein complexes bearing tyrosine phosphorylated YXXM motifs. Thus, the PI3K signaling pathway regulates the choice between epidermal cell differentiation and death at the cross-talk between tyrosine kinases and cadherin-associated catenins.

Fyn tyrosine kinase is a downstream mediator of Rho/PRK2 function in keratinocyte cell–cell adhesion

The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell–cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of β- and γ-catenin, which is associated with the establishment of keratinocyte cell–cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell–cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of β- and γ-catenin and p120ctn. The positive effects of activated Rho on cell–cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell–cell adhesion and induces tyrosine phosphorylation of β- and γ-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell–cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.

Citron Rho-interacting Kinase, a Novel Tissue-specific Ser/Thr Kinase Encompassing the Rho-Rac-binding Protein Citron

We have identified a novel serine/threonine kinase belonging to the myotonic dystrophy kinase family. The kinase can be produced in at least two different isoforms: a ∼240-kDa protein (Citron Rho-interacting kinase, CRIK), in which the kinase domain is followed by the sequence of Citron, a previously identified Rho/Rac binding protein; a ∼54-kDa protein (CRIK-short kinase (SK)), which consists mostly of the kinase domain. CRIK and CRIK-SK proteins are capable of phosphorylating exogenous substrates as well as of autophosphorylation, when tested by in vitro kinase assays after expression into COS7 cells. CRIK kinase activity is increased severalfold by coexpression of costitutively active Rho, while active Rac has more limited effects. Kinase activity of endogenous CRIK is indicated by in vitro kinase assays after immunoprecipitation with antibodies recognizing the Citron moiety of the protein. When expressed in keratinocytes, full-length CRIK, but not CRIK-SK, localizes into corpuscular cytoplasmic structures and elicits recruitment of actin into these structures. The previously reported Rho-associated kinases ROCK I and II are ubiquitously expressed. In contrast, CRIK exhibits a restricted pattern of expression, suggesting that this kinase may fulfill a more specialized function in specific cell types.

A PKC-η/Fyn-Dependent Pathway Leading to Keratinocyte Growth Arrest and Differentiation

Growth control of epithelial cells differs substantially from other cell types. Activation of Fyn, a Src kinase family member, is required for normal keratinocyte differentiation. We report that increased Fyn activity by itself suppresses growth of keratinocytes, but not dermal fibroblasts, through downmodulation of EGF receptor (EGFR) signaling. Protein kinase C-eta has also been implicated in keratinocyte growth/differentiation control. We show that growth suppression of keratinocytes by PKC-eta depends mostly on Fyn. PKC-eta activity is both necessary and sufficient for Fyn activation, PKC-eta and Fyn are found in association, and recombinant PKC-eta directly activates Fyn. Thus, our findings reveal a direct cross talk between PKC-eta and Fyn, which presides over the decision between keratinocyte (epithelial) cell growth and differentiation.

Syndecan-4: Dispensable or indispensable?

Studies examining the role of the cell-surface heparan sulfate proteoglycan syndecan-4 have yielded a plethora of information regarding its role in both cell-matrix and growth-factor mediated signaling events. Many of the initial conclusions drawn from such research placed syndecan-4 in a keystone position within various signaling pathways though the generation of syndecan-4 null mice have surprised many in the field by indicating otherwise. These contradictory results place researchers in the frustrating and yet exhilarating position of still asking the question, “What role does syndecan-4 play in life?” Published in 2003.

Rictor/mTORC2 deficiency enhances keratinocyte stress tolerance via mitohormesis.

How metabolic pathways required for epidermal tissue growth and remodeling influence the ability of keratinocytes to survive stressful conditions is still largely unknown. The mechanistic target of rapamycin complex 2 (mTORC2) regulates growth and metabolism of several tissues, but its functions in epidermal cells are poorly defined. Rictor is an adaptor protein essential for mTORC2 activity. To explore the roles of mTORC2 in the epidermis, we have conditionally deleted rictor in mice via K14-Cre-mediated homologous recombination and found that its deficiency causes moderate tissue hypoplasia, reduced keratinocyte proliferation and attenuated hyperplastic response to TPA. Noteworthy, rictor-deficient keratinocytes displayed increased lifespan, protection from senescence, and enhanced tolerance to cellular stressors such as growth factors deprivation, epirubicin and X-ray in vitro and radioresistance in vivo. Rictor-deficient keratinocytes exhibited changes in global gene expression profiles consistent with metabolic alterations and enhanced stress tolerance, a shift in cell catabolic processes from glycids and lipids to glutamine consumption and increased production of mitochondrial reactive oxygen species (ROS). Mechanistically, the resiliency of rictor-deficient epidermal cells relies on these ROS increases, indicating stress resistance via mitohormesis. Thus, our findings reveal a new link between metabolic changes and stress adaptation of keratinocytes centered on mTORC2 activity, with potential implications in skin aging and therapeutic resistance of epithelial tumors.

Tyrosine phosphorylation and SRC family kinases control keratinocyte cell-cell adhesion

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. We show here that β-catenin, γ-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of α-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of α-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of β- and γ-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn−/− mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of β-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo.