Biomedical subjects

E Calautti

Publications and source records attributed to E Calautti.

11 recordsLinked to original sources

PubMed2000

A PKC-eta/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.

Animals↗

PubMed1999

TGF-beta3, but not TGF-beta1, protects keratinocytes against 12-O-tetradecanoylphorbol-13-acetate-induced cell death in vitro and in vivo.

We have examined the role that individual TGF-beta isoforms, and in particular TGF-beta3, play in control of epidermal homeostasis. Mice with a knockout mutation of the TGF-beta3 gene die a few hours after birth. A full-thickness skin grafting approach was used to investigate the postnatal development and homeostatic control of the skin of these mice. Grafted skin of mice with a disruption of the TGF-beta3 gene developed similarly to grafts of wild type and TGF-beta1 knockout animals. However, a strikingly different response was observed after acute treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). When exposed to TPA, the grafted skin of wild type and TGF-beta1 knockout mice underwent a hyperplastic response similar to that of normal mouse skin. In marked contrast, TPA treatment of TGF-beta3 knockout grafts induced widespread areas of keratinocyte cell death. Analysis of cultured keratinocytes treated with purified TGF-beta isoforms revealed that TGF-beta3 plays a direct and specific function in protecting keratinocytes against TPA-induced cell death. The protective function of TGF-beta3 on TPA-induced cell death was not because of general suppression of the signaling pathways triggered by this agent, as ERK1/2 activation occurred to a similar if not greater extent in TGF-beta3-treated versus control keratinocytes. Instead, TGF-beta3 treatment led to a significant reduction in TPA-induced c-Jun N-terminal kinase activity, which was associated and possibly explained by specific counteracting effects of TGF-beta3 on TPA-induced disruption of keratinocyte focal adhesions.

Actins↗

PubMed1999

Cells shed from tumours show reduced clonogenicity, resistance to apoptosis, and in vivo tumorigenicity.

The goal of this study was to compare growth characteristics of cells shed from a tumour with the native tumour cells. The human colon adenocarcinoma LS174T and its highly metastatic subline LS LiM 6 were grown as tissue-isolated tumours in nude mice and perfused to collect shed cells. The tumours were then excised and prepared into single-cell suspensions. Clonogenicity in 0.3-0.9% agarose, apoptotic fraction, and in vivo tumorigenicity were determined for each population. In both tumour lines, shed cells were less clonogenic, more apoptotic and less tumorigenic than cells isolated directly from their native tissue. These findings suggest that shed cells have a low metastatic potential compared to native tumour cells, most likely because they represent an apoptotic population.

Adenocarcinoma↗

PubMed1998

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 approximately 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 approximately 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.

Amino Acid Sequence↗

PubMed1998

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 beta-catenin, gamma-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 alpha-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 alpha-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 beta- and gamma-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 beta-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.

Animals↗

PubMed1998

Inhibitory function of p21Cip1/WAF1 in differentiation of primary mouse keratinocytes independent of cell cycle control.

The cyclin-dependent kinase inhibitor p21(Cip1/WAF1) has been implicated as an inducer of differentiation. However, although expression of p21 is increased in postmitotic cells immediately adjacent to the proliferative compartment, its expression is decreased in cells further along the differentiation program. Expression of the p21 protein was decreased in terminally differentiated primary keratinocytes of mice, and this occurred by a proteasome-dependent pathway. Forced expression of p21 in these cells inhibited the expression of markers of terminal differentiation at both the protein and messenger RNA levels. These inhibitory effects on differentiation were not observed with a carboxyl-terminal truncation mutant or with the unrelated cyclin-dependent kinase inhibitor p16(INK4a), although all these molecules exerted similar inhibition of cell growth. These findings reveal an inhibitory role of p21 in the late stages of differentiation that does not result from the effects of p21 on the cell cycle.

Acetylcysteine↗

PubMed1997

A novel transmembrane semaphorin can bind c-src.

The semaphorins/collapsins constitute a family of genes unified by the presence of a "semaphorin domain" which has been conserved through metazoan evolution. The semaphorin family comprises both secreted and transmembrane molecules and is thought to be made up of ligands for as yet unidentified receptors. The functions are not known, with the exception of those of sema III (also referred as sem D and collapsin 1), D-sema I, and D-sema II, which have been shown to be involved in axonal pathfinding. Here report the identification of a mouse semaphorin cDNA, termed Sema VIb. Although Sema VIb contains the extracellular semaphorin domain, it lacks the immunoglobulin domain or thrombospondin repeats which are present in other described vertebrate (but not invertebrate) transmembrane semaphorins. During development Sema VIb mRNA is expressed in subregions of the nervous system and is particularly prominent in muscle. In adulthood, Sema VIb mRNA is expressed ubiquitously. The cytoplasmic domain of Sema VIb contains several proline-rich potential SH3 domain binding sites. Using an in vitro binding assay, we show that Sema VIb binds specifically the SH3 domain of the protooncogene c-src. In transfected COS cells Sema VIb coimmunoprecipitates with c-src. These results, along with our evidence that Sema VIb can form dimers, suggests that the semaphorin family not only serves as ligands but may include members, especially those which are transmembrane, which serve as receptors, triggering intracellular signaling via an src-related cascade.

Amino Acid Sequence↗

PubMed1995

fyn tyrosine kinase is involved in keratinocyte differentiation control.

Induction of tyrosine phosphorylation is an early and specific event which is required for mouse keratinocyte differentiation to occur, in response to both calcium and TPA (12-0-tetradecanoylphorbol-13-acetate). We report here that there is an increase of tyrosine kinase activity immunoprecipitable with anti-phosphotyrosine antibodies specifically in response to calcium--and a number of other divalent cations--within 2 min of exposure. Such an activity does not correspond to any of the known tyrosine kinases that were tested. A second tyrosine kinase activity is induced in response to both calcium and TPA, and has been identified as fyn, a nonreceptor tyrosine kinase of the src family. fyn activation is induced in keratinocytes within 6 hr of calcium exposure, but already within 2 min of TPA treatment. Cortactin, a p80-85 substrate of src- and fyn-related kinases that localizes with actin at cell adhesion sites, is increasingly tyrosine phosphorylated in calcium- and TPA-induced differentiation, with a time course which parallels that of fyn activation. Keratinocytes with a specific disruption of the fyn, but not yes kinase gene show no induction of phosphorylation of p80-85 proteins, and are significantly altered in their differentiation response both in vitro and in vivo. Thus, at least two tyrosine kinase activities are induced in keratinocyte differentiation, one of which has been identified as fyn and shown to be specifically involved in this process.

Animals↗

PubMed1995

Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation.

The mechanism of cell cycle withdrawal during terminal differentiation is poorly understood. We report here that the cyclin-dependent kinase (CDK) inhibitor p21Cip1/WAF1 is induced at early times of both keratinocyte and myoblast differentiation. p21Cip1/WAF1 induction is accompanied by a drastic inhibition of total Cdk2, as well as p21Cip1/WAF1-associated CDK kinase activities. p21Cip1/WAF1 has been implicated in p53-mediated G1 arrest and apoptosis. In keratinocyte differentiation, Cip1/WAF1 induction is observed even in cells derived from p53-null mice. Similarly, keratinocyte differentiation is associated with induction of Cip1/WAF1 promoter activity in both wild-type and p53-negative keratinocytes. Induction of the Cip1/WAF1 promoter upon differentiation is abolished by expression of an adenovirus E1A oncoprotein (d1922/947), which is unable to bind p105-Rb, p107, or cyclin A but which still binds the nuclear phosphoprotein p300. Overexpression of p300 can suppress the E1A effect, independent of its direct binding to E1A. Thus, terminal differentiation-induced growth arrest in both keratinocyte and myoblast systems is associated with induction of Cip1/WAF1 expression. During keratinocyte differentiation, Cip1/WAF1 induction does not require p53 but depends on the transcriptional modulator p300.

Animals↗

PubMed1994

Functional evidence for an extracellular calcium receptor mechanism triggering tyrosine kinase activation associated with mouse keratinocyte differentiation.

Calcium-induced keratinocyte differentiation is associated with tyrosine phosphorylation of a p62 protein which associates with the ras-GTPase activating protein (GAP). We have examined the nature of the calcium signal triggering p62 phosphorylation. EGTA, a specific chelator of calcium, was able to completely block calcium-induced p62 phosphorylation, even after using conditioned medium from calcium-treated keratinocytes. Preventing calcium-induced cell-cell contacts by anti-cadherin antibodies did not inhibit tyrosine phosphorylation. Slight increases in extracellular calcium concentrations (0.15 or 0.30 mM) were already sufficient to induce p62 phosphorylation. Other divalent cations, such as magnesium, zinc, nickel, and cobalt, but not the trivalent cation lanthanum, induced p62 phosphorylation to a similar extent as calcium. There was no close correlation between the ability of the various ions to induce p62 phosphorylation and increase free intracellular calcium. Similarly, treatment of primary keratinocytes with the calcium ionophores A23187 or X537A did not induce p62 phosphorylation, although it increased free intracellular calcium levels. Finally, blockers of potassium uptake, which is induced by calcium, did not inhibit p62 phosphorylation. Thus, in keratinocyte differentiation, calcium is likely to provide the primary signal for p62 tyrosine phosphorylation and may act directly at the cell membrane through a "cationic receptor mechanism" analogous to that described in other cell types.

Animals↗

PubMed1992

Specific changes of Ras GTPase-activating protein (GAP) and a GAP-associated p62 protein during calcium-induced keratinocyte differentiation.

Induction of tyrosine phosphorylation occurs as an early and specific event in keratinocyte differentiation. A set of tyrosine-phosphorylated substrates which transduce mitogenic signals by tyrosine kinases has previously been identified. We show here that of these substrates, the Ras GTPase-activating protein, GAP, is specifically affected during calcium-induced keratinocyte differentiation. As early as 10 min after calcium addition to cultured primary mouse keratinocytes, GAP associates with tyrosine-phosphorylated proteins and translocates to the membrane. In addition, a GAP-associated protein of approximately 62 kDa (p62) becomes rapidly and heavily tyrosine phosphorylated in both membrane and cytosolic fractions. This protein corresponds to the major tyrosine-phosphorylated protein that is induced in differentiating keratinocytes as early as 5 min after calcium addition. p62 phosphorylation was not observed after exposure of these cells to epidermal growth factor, phorbol ester, or transforming growth factor beta. In contrast, PLC gamma and P13K were tyrosine phosphorylated after epidermal growth factor, but not calcium, stimulation. Thus, changes of Ras GAP and an associated p62 protein occur as early and specific events in keratinocyte differentiation and appear to involve a calcium-induced tyrosine kinase.

3T3 Cells↗