Biomedical subjects

Lina Dagnino

Publications and source records attributed to Lina Dagnino.

15 recordsLinked to original sources

PubMed2006

Differentiation and injury-repair signals modulate the interaction of E2F and pRB proteins with novel target genes in keratinocytes.

E2F transcription factors are central to epidermal morphogenesis and regeneration after injury. The precise nature of E2F target genes involved in epidermal formation and repair has yet to be determined. Identification of these genes is essential to understand how E2F proteins regulate fundamental aspects of epidermal homeostasis and transformation. We have conducted a genome-wide screen using CpG island microarray analysis to identify novel promoters bound by E2F3 and E2F5 in human keratinocytes. We further characterized several of these genes, and determined that multiple E2F and retinoblastoma (pRb) family proteins associate with them in exponentially proliferating cells. We also assessed the effect on E2F and pRb binding to those genes in response to differentiation induced by bone morphogenetic protein-6 (BMP-6), or to activation of repair mechanisms induced by transforming growth factor-beta (TGF-beta). These studies demonstrate promoter- and cytokine-specific changes in binding profiles of E2F and/or pRb family proteins. For example, E2F1, 3, 4 and p107 were recruited to the N-myc promoter in cells treated with BMP-6, whereas E2F1, 3, 4, 5, p107 and p130 were bound to this promoter in the presence of TGF-beta. Functionally, these different interactions resulted in transcriptional repression by BMP-6 and TGF-beta of the N-myc gene, via mechanisms that involved E2F binding to the promoter and association with pRb-family proteins. Thus, multiple combinations of E2F and pRb family proteins may associate with and transcriptionally regulate a given target promoter in response to differentiation and injury-repair stimuli in epidermal keratinocytes.

Animals↗

PubMed2005

RGS14 is a microtubule-associated protein.

Heterotrimeric G-proteins and their regulators are emerging as important players in modulating microtubule polymerization dynamics and in spindle force generation during cell division in C. elegans, D. melanogaster and mammals. We recently demonstrated that RGS14 is required for completion of the first mitotic division of the mouse embryo, and that it regulates microtubule organization in vivo. Here, we demonstrate that RGS14 is a microtubule-associated protein and a component of the mitotic spindle that may regulate microtubule polymerization and spindle organization. Taxol-stabilized tubulin, but not depolymerized tubulin coimmunoprecipitates with RGS14 from cell extracts. Furthermore, RGS14 copurifies with tubulin from porcine brain following multiple rounds of microtubule polymerization/depolymerization and binds directly to microtubules formed in vitro from pure tubulin (KD = 1.3 +/- 0.3 microM). Both RGS14 and Galpha(i1) in the presence of exogenous GTP promote tubulin polymerization, which is dependent on additional microtubule-associated proteins. However, preincubation of RGS14 with Galpha(i1)-GDP precludes either from promoting microtubule polymerization, suggesting that a functional GTP/GDP cycle is necessary. Finally, we show that RGS14 is a component of mitotic asters formed in vitro from HeLa cell extracts and that depletion of RGS14 from cell extracts blocks aster formation. Collectively, these results show that RGS14 is a microtubule-associated protein that may modulate microtubule dynamics and spindle formation.

Animals↗

PubMed2005

A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid ( 6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

Adherens Junctions↗

PubMed2005

Signalling in the epidermis: the E2F cell cycle regulatory pathway in epidermal morphogenesis, regeneration and transformation.

The epidermis is the outermost layer in the skin, and it is the first line of defence against the environment. The epidermis also provides a barrier against loss of fluids and electrolytes, which is crucial for life. Essential in the maintenance of this tissue is its ability to continually self-renew and regenerate after injury. These two characteristics are critically dependent on the ability of the principal epidermal cell type, the keratinocyte, to proliferate and to respond to differentiation cues. Indeed, the epidermis is a multilayered tissue composed of keratinocyte stem cells and their differentiated progeny. Central for the control of cell proliferation is the E2F transcription factor regulatory network. This signaling network also includes cyclins, cdk, cdk inhibitors and the retinoblastoma (pRb) family of proteins. The biological importance of the E2F/pRb pathway is emphasized by the fact that a majority of human tumours exhibit alterations that disrupt the ability of pRb proteins to inhibit E2F, leading to permanent activation of the latter. Further, E2F is essential for normal epidermal regeneration after injury. Other member of the E2F signaling pathway are also involved in epidermal development and pathophysiology. Thus, whereas the pRb family of proteins is essential for epidermal morphogenesis, abnormal regulation of cyclins and E2F proteins results in tumorgenesis in this tissue. In this review, we discuss the role of each member of this important growth regulatory network in epidermal formation, homeostasis and carcinogenesis.

Animals↗

PubMed2005

Differential expression of regulator of G-protein signaling R12 subfamily members during mouse development.

Regulators of G-protein Signaling (RGS proteins) are a multigene family of GTPase-accelerating proteins for the Galpha subunit of heterotrimeric G-proteins. The mammalian R12 RGS protein subfamily is composed of RGS12 and RGS14, two proteins characterized by their multidomain architecture of hallmark RGS domain, tandem Ras-binding domains (RBDs), and a second Galpha interacting domain, the GoLoco motif. The Rgs12 gene generates multiple splice variants, the largest of which encodes N-terminal PDZ and PTB domains in addition to the core RGS/RBD/GoLoco motifs. The Rgs14 gene encodes a protein similar to the non-PDZ/PTB domain RGS12 splice variants. The spatiotemporal expression patterns of RGS12 and RGS14 proteins were examined by immunohistochemistry in a developmental series of postimplantation mouse embryo. We report that RGS12 splice variants exhibit differential spatiotemporal patterns of expression during postimplantation embryogenesis, suggesting nonoverlapping roles. In contrast, RGS14 is found ubiquitously throughout the postimplantation period. We conclude that R12 subfamily RGS proteins likely play significant and different roles in specific tissues and periods of mouse embryogenesis.

Alternative Splicing↗

PubMed2005

Shh controls epithelial proliferation via independent pathways that converge on N-Myc.

Shh signaling induces proliferation of many cell types during development and disease, but how Gli transcription factors regulate these mitogenic responses remains unclear. By genetically altering levels of Gli activator and repressor functions in mice, we have demonstrated that both Gli functions are involved in the transcriptional control of N-myc and Cyclin D2 during embryonic hair follicle development. Our results also indicate that additional Gli-activator-dependent functions are required for robust mitogenic responses in regions of high Shh signaling. Through posttranscriptional mechanisms, including inhibition of GSK3-beta activity, Shh signaling leads to spatially restricted accumulation of N-myc and coordinated cell cycle progression. Furthermore, a temporal shift in the regulation of GSK3-beta activity occurs during embryonic hair follicle development, resulting in a synergy with beta-catenin signaling to promote coordinated proliferation. These findings demonstrate that Shh signaling controls the rapid and patterned expansion of epithelial progenitors through convergent Gli-mediated regulation.

Animals↗

PubMed2005

A cyclosporine-sensitive psoriasis-like disease produced in Tie2 transgenic mice.

Psoriasis is a common, persistent skin disorder characterized by recurrent erythematous lesions thought to arise as a result of inflammatory cell infiltration and activation of keratinocyte proliferation. Unscheduled angiogenic growth has also been proposed to mediate the pathogenesis of psoriasis although the cellular and molecular basis for this response remains unclear. Recently, a role for the angiopoietin signaling system in psoriasis has been suggested by studies that demonstrate an up-regulation of the tyrosine kinase receptor Tie2 (also known as Tek) as well as angiopoietin-1 and angiopoietin-2 in human psoriatic lesions. To examine temporal expression of Tie2, we have developed a binary transgenic approach whereby expression of Tie2 can be conditionally regulated by the presence of tetracycline analogs in double-transgenic mice. A psoriasis-like phenotype developed in double-transgenic animals within 5 days of birth and persisted throughout adulthood. The skin of affected mice exhibited many cardinal features of human psoriasis including epidermal hyperplasia, inflammatory cell accumulation, and altered dermal angiogenesis. These skin abnormalities resolved completely with tetracycline-mediated suppression of transgene expression, thereby illustrating a complete dependence on Tie2 signaling for disease maintenance and progression. Furthermore, the skin lesions in double-transgenic mice markedly improved after administration of the immunosuppressive anti-psoriatic agent cyclosporine, thus demonstrating the clinical significance of this new model.

Angiopoietins↗

PubMed2005

Analysis of E2F factors during epidermal differentiation.

The multigene E2F family of transcription factors is central in the control of cell cycle progression. The expression and activity of E2F proteins is tightly regulated transcriptionally and posttranslationally as a function of the proliferation and differentiation status of the cell. In this chapter, we review protocols designed to determine E2F mRNA abundance in tissues by in situ hybridization techniques. The ability to culture primary epidermal keratinocytes and maintain them as either undifferentiated or terminally differentiated cells allows the biochemical and molecular characterization of changes in E2F expression and activity. Thus, we also discuss in detail methods to analyze E2F protein abundance by immunoblot and their ability to bind DNA in cultured cells using electrophoretic mobility shift assays.

Animals↗

PubMed2004

The DP-1 transcription factor is required for keratinocyte growth and epidermal stratification.

The epidermis is a stratified epithelium constantly replenished through the ability of keratinocytes in its basal layer to proliferate and self-renew. The epidermis arises from a single-cell layer ectoderm during embryogenesis. Large proliferative capacity is central to ectodermal cell and basal keratinocyte function. DP-1, a heterodimeric partner of E2F transcription factors, is highly expressed in the ectoderm and all epidermal layers during embryogenesis. To investigate the role of DP-1 in epidermal morphogenesis, we inhibited DP-1 activity through exogenous expression of a dominant-negative mutant (dnDP-1). Expression of the dnDP-1 mutant interferes with binding of E2F/DP-1 heterodimers to DNA and inhibits DNA replication, as well as cyclin A mRNA and protein expression. Chromatin immunoprecipitation analysis demonstrated that the cyclin A promoter is predominantly bound in proliferating keratinocytes by complexes containing E2F-3 and E2F-4. Thus, the mechanisms of decreased expression of cyclin A in the presence of dnDP-1 seem to involve inactivation of DP-1 complexes containing E2F-3 and E2F-4. To assess the consequences on epidermal morphogenesis of inhibiting DP-1 activity, we expressed dnDP-1 in rat epithelial keratinocytes in organotypic culture and observed that DP-1 inhibition negatively affected stratification of these cells. Likewise, expression of dnDP-1 in embryonic ectoderm explants produced extensive disorganization of subsequently formed epidermal basal and suprabasal layers, interfering with normal epidermal formation. We conclude that DP-1 activity is required for normal epidermal morphogenesis and ectoderm-to-epidermis transition.

Adenoviridae↗

PubMed2004

RGS14 is a mitotic spindle protein essential from the first division of the mammalian zygote.

Heterotrimeric G protein alpha subunits, RGS proteins, and GoLoco motif proteins have been recently implicated in the control of mitotic spindle dynamics in C. elegans and D. melanogaster. Here we show that "regulator of G protein signaling-14" (RGS14) is expressed by the mouse embryonic genome immediately prior to the first mitosis, where it colocalizes with the anastral mitotic apparatus of the mouse zygote. Loss of Rgs14 expression in the mouse zygote results in cytofragmentation and failure to progress to the 2-cell stage. RGS14 is found in all tissues and segregates to the nucleus in interphase and to the mitotic spindle and centrioles during mitosis. Alteration of RGS14 levels in exponentially proliferating cells leads to cell growth arrest. Our results indicate that RGS14 is one of the earliest essential product of the mammalian embryonic genome yet described and has a general role in mitosis.

Animals↗

PubMed2004

Analysis of interactions between regulator of G-protein signaling-14 and microtubules.

Microtubules are dynamic polymers essential for mitosis and cell division, intracellular transport, and maintaining cell organization and structure. Microtubule dynamics are tightly controlled in a context-specific manner by a myriad of microtubule-associated proteins. We have identified regulator of G-protein signaling-14 (RGS14) as a microtubule-associated protein. RGS14 is a component of the mitotic apparatus that binds directly to and stabilizes microtubules in vitro and is essential for the first cell division in the mouse embryo. This article describes methods used for examining the impact of the RGS14/microtubule interaction in vivo and in vitro.

Animals↗

PubMed2003

Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

Animals↗

PubMed2002

Active nuclear import and export pathways regulate E2F-5 subcellular localization.

Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.

Active Transport, Cell Nucleus↗

PubMed2002

E2F-1 is essential for normal epidermal wound repair.

E2F factors are involved in proliferation and apoptosis. To understand the role of E2F-1 in the epidermis, we screened wild type and E2F-1(-/-) keratinocyte mRNA for genes differentially expressed in the two cell populations. We demonstrate the reduced expression of integrins alpha(5), alpha(6), beta(1), and beta(4) in E2F-1(-/-) keratinocytes associated with reduced activation of Jun terminal kinase and Erk upon integrin stimulation. As a consequence of altered integrin expression and function, E2F-1(-/-) keratinocytes also show impaired migration, adhesion to extracellular matrix proteins, and a blunted chemotactic response to transforming growth factor-gamma1. E2F-1(-/-) keratinocytes, but not dermal fibroblasts, exhibit altered patterns of proliferation, including significant delays in transit through both G(1) and S phases of the cell cycle. Recognizing that proliferation and migration are key for proper wound healing in vivo, we postulated that E2F-1(-/-) mice may exhibit abnormal epidermal repair upon injury. Consistent with our hypothesis, E2F-1(-/-) mice exhibited impaired cutaneous wound healing. This defect is associated with substantially reduced local inflammatory responses and rates of re-epithelialization. Thus, we demonstrate that E2F-1 is indispensable for a hitherto unidentified cell type-specific and unique role in keratinocyte proliferation, adhesion, and migration as well as in proper wound repair and epidermal regeneration in vivo.

Animals↗

PubMedDate not supplied

Delayed apoptosis post-cadmium injury in renal proximal tubule epithelial cells.

BACKGROUND: Accumulation of the widespread environmental toxin cadmium (Cd) in the kidney results initially in proximal tubule dysfunction. Exposure to Cd has been previously shown to induce apoptosis in LLC-PK (Lily Laboratory Culture, Porcine Kidney) cells, which are a model of proximal tubule epithelium. HYPOTHESIS: We postulated that modulation of the components of the apoptotic pathway triggered by Cd is amenable to therapeutic intervention. METHODS: We subjected confluent LLC-PK cells grown on two-compartment filters and on plastic to Cd (1-50 microM). Apoptosis and changes in components of the apoptotic pathway were measured by immunocytochemical and immunoblot analysis during the period of exposure and following Cd withdrawal. RESULTS: Insignificant apoptosis was seen during exposure to Cd and immediately after removal of this metal. Two waves of apoptosis were noted 6 and 48 h after the Cd was removed from the apical compartment. The apoptosis 48 h post-Cd exposure was accompanied by a decrease in cellular ATP levels and transepithelial resistance and preceded by an increase in p38 phosphorylation. Inhibition of p38 mitogen-activated protein kinase activity decreased the delayed apoptotic peak, without affecting the rate of recovery of the integrity of the renal epithelium. IGF-1 neither altered the delayed apoptosis nor facilitated the rate of recovery of the integrity of the renal epithelium. CONCLUSION: We demonstrate that following exposure to Cd, renal epithelial cells undergo significant apoptosis, which appears to involve p38 and is not amenable to IGF therapy.

Animals↗