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Biomedical subjects

G P Dotto

Publications and source records attributed to G P Dotto.

At least 19 recordsLinked to original sources
PubMed2001

Cross talk among calcineurin, Sp1/Sp3, and NFAT in control of p21(WAF1/CIP1) expression in keratinocyte differentiation.

Calcium functions as a trigger for the switch between epithelial cell growth and differentiation. We report here that the calcium/calmodulin-dependent phosphatase calcineurin is involved in this process. Treatment of primary mouse keratinocytes with cyclosporin A, an inhibitor of calcineurin activity, suppresses the expression of terminal differentiation markers and of p21(WAF1/Cip1) and p27(KIP1), two cyclin-dependent kinase inhibitors that are usually induced with differentiation. In parallel with down-modulation of the endogenous genes, suppression of calcineurin function blocks induction of the promoters for the p21(WAF1/Cip1) and loricrin differentiation marker genes, whereas activity of these promoters is enhanced by calcineurin overexpression. The calcineurin- responsive region of the p21 promoter maps to a 78-bp Sp1/Sp3-binding sequence next to the TATA box, and calcineurin induces activity of the p21 promoter through Sp1/Sp3-dependent transcription. We find that the endogenous NFAT-1 and -2 transcription factors, major downstream targets of calcineurin, associate with Sp1 in keratinocytes in a calcineurin-dependent manner, and calcineurin up-regulates Sp1/Sp3-dependent transcription and p21 promoter activity in synergism with NFAT1/2. Thus, our study reveals an important role for calcineurin in control of keratinocyte differentiation and p21 expression, and points to a so-far-unsuspected interconnection among this phosphatase, NFATs, and Sp1/Sp3-dependent transcription.

Animals↗
PubMed2001

Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation.

The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. We show that keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1 expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation.

Animals↗
PubMed2000

Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis.

Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K-/- mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with depletion of specific neuronal populations. These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Our results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS.

Animals↗
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↗
PubMed2000

Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo.

The regression phase of the hair cycle (catagen) is an apoptosis-driven process accompanied by terminal differentiation, proteolysis, and matrix remodeling. As an inhibitor of keratinocyte proliferation and inductor of keratinocyte apoptosis, transforming growth factor beta1 (TGF-beta1) has been proposed to play an important role in catagen regulation. This is suggested, for example, by maximal expression of TGF-beta1 and its receptors during late anagen and the onset of catagen of the hair cycle. We examined the potential involvement of TGF-beta1 in catagen control. We compared the first spontaneous entry of hair follicles into catagen between TGF-beta1 null mice and age-matched wild-type littermates, and assessed the effects of TGF-beta1 injection on murine anagen hair follicles in vivo. At day 18 p.p., hair follicles in TGF-beta1 -/- mice were still in early catagen, whereas hair follicles of +/+ littermates had already entered the subsequent resting phase (telogen). TGF-beta1-/- mice displayed more Ki-67-positive cells and fewer apoptotic cells than comparable catagen follicles from +/+ mice. In contrast, injection of TGF-beta1 into the back skin of mice induced premature catagen development. In addition, the number of proliferating follicle keratinocytes was reduced and the number of TUNEL + cells was increased in the TGF-beta1-treated mice compared to controls. Double visualization of TGF-beta type II receptor (TGFRII) and TUNEL reactivity revealed colocalization of apoptotic nuclei and TGFRII in catagen follicles. These data strongly support that TGF-beta1 ranks among the elusive endogenous regulators of catagen induction in vivo, possibly via the inhibition of keratinocyte proliferation and induction of apoptosis. Thus, TGF-betaRII agonists and antagonists may provide useful therapeutic tools for human hair growth disorders based on premature or retarded catagen development (effluvium, alopecia, hirsutism).

Animals↗
PubMed1999

The TGF-beta2 isoform is both a required and sufficient inducer of murine hair follicle morphogenesis.

Hair follicle development serves as an excellent model to study control of organ morphogenesis. Three specific isoforms of TGF-beta exist which exhibit a distinct pattern of expression during hair follicle morphogenesis. To clarify the still elusive role of these factors in hair follicle development, we have used a combined genetic and functional approach: analysis of hair follicle development in mice with disruptions of the TGF-beta1, 2, and 3 genes was coupled with a direct functional test of the effect of added purified factors on fetal hair follicle development in skin organ cultures. TGF-beta2 null mice exhibited a profound delay of hair follicle morphogenesis, with a 50% reduced number of hair follicles. In contrast to hair follicle development, growth and differentiation of interfollicular keratinocytes proceeded unimpaired. Unlike TGF-beta2-/- mice, mice with a disruption of the TGF-beta1 gene showed slightly advanced hair follicle formation, while lack of the TGF-beta3 gene did not have any effects. Treatment of wild-type, embryonic skin explants (E14.5 or E15.5) with TGF-beta2 protein in either soluble form or slow release beads induced hair follicle development and epidermal hyperplasia, while similar TGF-beta1 treatment exerted suppressive effects. Thus, the TGF-beta2 isoform plays a specific role, not shared by the other TGF-beta isoforms, as an inducer of hair follicle morphogenesis and is both required and sufficient to promote this process.

Animals↗
PubMed1999

p21(WAF1/Cip1) functions as a suppressor of malignant skin tumor formation and a determinant of keratinocyte stem-cell potential.

p21(WAF1/Cip1) is one of the best characterized downstream targets of p53, and the growth suppressing function of this cyclin-dependent kinase inhibitor is well established. However, whether p21 exerts a tumor-suppressing function of its own remains to be established. We report here that, similarly to loss of p53, disruption of the p21(WAF1/Cip1) gene results in a markedly increased susceptibility to chemically induced skin carcinoma formation, whereas the number of papillomas is reduced. Previous evidence indicates that malignant versus benign keratinocyte tumor formation is likely to involve distinct target-cell populations with a different commitment to differentiation. In parallel with the increased susceptibility to carcinoma formation, loss of p21(WAF1/Cip1) was found to promote keratinocyte subpopulations with increased growth/differentiation potential, including clonal growth capability, reversible commitment to differentiation, and capability to generate all types of terminally differentiated keratinocytes present in vivo, not only in the interfollicular epidermis but also in hair follicles. Thus, these findings have revealed a function of p21 as a suppressor of malignant but not benign skin-tumor formation and a determinant of the growth/differentiation potential of keratinocyte subpopulations.

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

Signal transduction pathways controlling the switch between keratinocyte growth and differentiation.

Self-renewing epithelia are characterized by a high turnover rate and a fine balance between growth and differentiation. Such a balance is influenced by many exogenous factors, including gradients of diffusible molecules, cell/substrate adhesion contacts, and direct cell-cell communication. The inter-connection between these various extracellular signals and underlying intracellular pathways is clearly of great interest. Primary keratinocytes of either human or murine origin provide an ideal experimental system to elucidate early signaling events involved in the control of epithelial differentiation. Relative to established cell lines, use of a primary system eliminates the possibility of alterations in critical regulatory events which may occur during prolonged propagation in culture. Primary keratinocytes are easily grown in large numbers, and their differentiation can be induced under well-defined culture conditions. The ensuing rapid and homogeneous response is amenable to careful biochemical analysis. Gene transfer technology (transient transfections, adenoviral and retroviral vectors), together with the use of keratinocytes derived from gene knockout and transgenic mice, makes it possible to assess the specific contribution of individual genes to the control of the differentiation process. This review focuses on the significant progress that has been made over the last few years in our understanding of the specific signals that trigger keratinocyte differentiation, the underlying signaling pathways, and how they impinge on specific transcription and cell-cycle control mechanisms associated with the onset of keratinocyte differentiation. Recent developments and future directions in this important area of research will be highlighted.

Animals↗
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

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↗
PubMed1998

Differentiation-specific increase in ALA-induced protoporphyrin IX accumulation in primary mouse keratinocytes.

A treatment regimen that takes advantage of the induction of intracellular porphyrins such as protoporphyrin IX (PPIX) by exposure to exogenous 5-amino-laevulinic acid (ALA) followed by localized exposure to visible light represents a promising new approach to photodynamic therapy (PDT). Acting upon the suggestion that the effectiveness of ALA-dependent PDT may depend upon the state of cellular differentiation, we investigated the effect of terminal differentiation upon ALA-induced synthesis of and the subsequent phototoxicity attributable to PPIX in primary mouse keratinocytes. Induction of keratinocyte differentiation augmented intracellular PPIX accumulation in cells treated with ALA. These elevated PPIX levels resulted in an enhanced lethal photodynamic sensitization of differentiated cells. The differentiation-dependent increase in cellular PPIX levels resulted from several factors including: (a) increased ALA uptake, (b) enhanced PPIX production and (c) decreased PPIX export into the culture media. Simultaneously, steady-state levels of coproporphyrinogen oxidase mRNA increased but aminolaevulinic acid dehydratase mRNA levels remained unchanged. From experiments using 12-o-tetradecanoylphorbol-13-acetate, transforming growth factor beta 1 and calcimycin we demonstrated that the increase in PPIX concentration in terminally differentiating keratinocytes is calcium- and differentiation specific. Stimulation of the haem synthetic capacity is seen in primary keratinocytes, but not in PAM 212 cells that fail to undergo differentiation. Interestingly, increased PPIX formation and elevated coproporphyrinogen oxidase mRNA levels are not limited to differentiating keratinocytes; these were also elevated in the C2C12 myoblast and the PC12 adrenal cell lines upon induction of differentiation. Overall, the therapeutic implications of these results are that the effectiveness of ALA-dependent PDT depends on the differentiation status of the cell and that this may enable selective targeting of several tissue types.

Aminolevulinic Acid↗
PubMed1997

Involvement of the Sp3 transcription factor in induction of p21Cip1/WAF1 in keratinocyte differentiation.

The cyclin-dependent kinase inhibitor p21 is induced in several in vitro terminal differentiation systems as well as in differentiating tissues in vivo. To determine the mechanism responsible for p21 induction during differentiation of mouse primary keratinocytes, we performed a deletion analysis of the p21 promoter. The minimal region of the p21 promoter required for its induction in keratinocyte differentiation consists of a contiguous stretch of 78 base pairs, which contains a GC-rich region as well as the TATA box. We determined that transcription factors Sp1 and Sp3, present in primary keratinocyte nuclear extracts, bind the GC region concomitantly. Expression studies established that both Sp1 and Sp3 activate the p21 promoter, but showed that only Sp3 overexpression enhances promoter inducibility during differentiation. Furthermore, disruption of the GC-rich region dramatically decreases transcription factor binding as well as promoter activity and inducibility upon differentiation. The overexpression of either Sp1 or Sp3 restores the basal activity of the disrupted promoter, but only Sp3 can restore its inducibility. These findings show that both Sp1 and Sp3 can contribute to the basal activity of the p21 promoter, and establish Sp3 as a specific transcription factor involved in the induction of p21 promoter during keratinocyte differentiation.

Animals↗
PubMed1997

Primary mouse keratinocyte cultures contain hair follicle progenitor cells with multiple differentiation potential.

The interfollicular epidermis contains a single type of terminally differentiated keratinocytes, whereas hair follicles are composed of a minimum of six or seven distinct types. Whether or not these various populations of terminally differentiated keratinocytes originate from one or more progenitor cells has not been established. A related and important question is whether keratinocyte progenitor cells with a pluripotent potential, able to form not only epidermis but also hair follicles, can be maintained in vitro for any period of time. We have addressed these questions using skin reconstitution assays with admixed populations of genetically labeled, cultured keratinocytes. Examination of reconstituted epidermis and hair follicles showed that neither was composed of a random mixture of differently labeled keratinocytes, as would be predicted if they originated from a random reassociation of cells. Instead, the reconstituted interfollicular epidermis contained distinct columnar units, comprising all the overlying layers and most likely derived from a single progenitor cell. In contrast, hair follicles were found to be composed of cells of multiple origin, with each population showing a striking localization to a separate concentric region. The vast majority of reconstituted follicles appeared to derive from a minimum of two or, in a significant fraction of cases, three progenitor cells, one for the generation of the shaft (cuticle, cortex, and medulla), one for the inner root sheath, and the third for the outer root sheath. The general implications of these findings for epidermis and hair follicle formation and for keratinocyte stem cell cultivation are discussed.

Animals↗
PubMed1996

The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.

p21Cip1/WAF1 was the first cyclin-dependent kinase (CDK) inhibitor to be identified, as a mediator of p53 in DNA damage-induced growth arrest, cell senescence, and direct CDK regulation. p21 may also play an important role in differentiation-associated growth arrest, as its expression is augmented in many terminally differentiating cells. A general involvement of p21 in growth/differentiation control and tumor suppression has been questioned, as mice lacking p21 undergo a normal development, harbor no gross alterations in any of their organs, and exhibit no increase in spontaneous tumor development. However, a significant imbalance between growth and differentiation could be unmasked under conditions where normal homeostatic mechanisms are impaired. We report here that primary keratinocytes derived from p21 knockout mice, transformed with a ras oncogene, and injected subcutaneously into nude mice exhibit a very aggressive tumorigenic behavior, which is not observed with wild-type control keratinocytes nor with keratinocytes with a disruption of the closely related p27 gene. p21 knockout keratinocytes tested under well-defined in vitro conditions show a significantly increased proliferative potential, which is also observed but to a lesser extent with p27 knockout cells. More profound differences were found in the differentiation behavior of p21 versus p27 knockout keratinocytes, with p21 (but not p27) deficiency causing a drastic down-modulation of differentiation markers linked with the late stages of the keratinocyte terminal differentiation program. Thus, our results reveal a so far undetected role of p21 in tumor suppression, demonstrate that this function is specific as it cannot be attributed to the closely related p27 molecule, and point to an essential involvement of p21 in terminal differentiation control, which may account for its role in tumor suppression.

Animals↗
PubMed1996

The product of the mouse nude locus, Whn, regulates the balance between epithelial cell growth and differentiation.

Mutations in the winged-helix nude (whn) gene result in the nude mouse and rat phenotypes. The pleiotropic nude phenotype which affects the hair, skin, and thymus suggests that whn plays a pivotal role in the development and/or maintenance of these organs. However, little is known about whn function in these organs. We show here that in skin whn is specifically expressed in epithelial cells and not the mesenchymal cells, and using a hair reconstitution assay, we demonstrate that the abnormal nude mouse hair development is attributable to a functional defect of the epithelial cells. Examination of nude mouse primary keratinocytes in culture revealed that these cells have an increased propensity to differentiate in an abnormal fashion, even under conditions that promote proliferation. Furthermore, nude mouse keratinocytes displayed a 100-fold increased sensitivity to the growth-inhibitory/differentiation effects of the phorbol ester TPA. In parallel with these findings, we directly show that whn functions as a transcription factor that can specifically suppress expression of differentiation/TPA-responsive genes. The region of Whn responsible for these effects was mapped to the carboxy-terminal transactivating domain. These results establish whn as a key regulatory factor involved in maintaining the balance between keratinocyte growth and differentiation. The general implications of these findings for an epithelial self-renewal model will be discussed.

Animals↗
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↗