Biomedical subjects

Kursad Turksen

Publications and source records attributed to Kursad Turksen.

13 recordsLinked to original sources

PubMed2006

The temporal and spatial expression of Claudins in epidermal development and the accelerated program of epidermal differentiation in K14-CaSR transgenic mice.

The importance of the epidermal permeability barrier (EPB) in protecting the mammalian species against harmful UV irradiation, microorganism invasion and water loss is well recognized, as is the role of calcium (Ca(2+)) in keratinocyte differentiation, cell-cell contact and the EPB. In a previous study, we reported that the overexpression of the Ca(2+)-sensing receptor (CaSR) in the undifferentiated basal cells of the epidermis induced a modified epidermal differentiation program including an accelerated EPB formation in transgenic mice, suggesting a role for CaSR signaling in the differentiation of embryonic epidermal cells during development. We now describe the expression profile of claudins (Cldns) and keratin markers in the accelerated EPB formation of K14-CaSR transgenic mice during development as compared to the wild type from E12.5 to newborn stages. Our data show that the transgenic epidermis undergoes an advanced epidermal differentiation program as compared to the wild type as evidenced morphologically as well as by the expression of K14, K1, loricrin, Cldn6, Cldn18 and Cldn11. In addition, we report for the first time the sequential expression of Cldns in epidermal development and describe that the localization of some Cldns change within the epidermis as it matures. Furthermore, we demonstrate that Cldn6 is expressed very early in epidermal morphogenesis, followed by Cldn18, Cldn11 and Cldn1.

Animals↗

PubMed2006

Role of the Cldn6 cytoplasmic tail domain in membrane targeting and epidermal differentiation in vivo.

It is widely recognized that the claudin (Cldn) family of four tetraspan transmembrane proteins is crucial for tight junction assembly and permeability barrier function; however, the precise role of the tail and loop domains in Cldn function is not understood. We hypothesized that the cytoplasmic tail domain of Cldn6 is crucial for membrane targeting and hence epidermal permeability barrier (EPB) formation. To test this hypothesis via a structure-function approach, we generated a tail deletion of Cldn6 (CDelta187) and evaluated its role in epidermal differentiation and EPB formation through its forced expression via the involucrin (Inv) promoter in the suprabasal compartment of the transgenic mouse epidermis. Even though a functional barrier formed, Inv-CDelta187 mice displayed histological and biochemical abnormalities in the epidermal differentiation program and stimulation of epidermal cell proliferation in both the basal and suprabasal compartments of the interfolliclar epidermis, leading to a thickening of the epidermis after 1 week of age that persisted throughout life. Although some membrane localization was evident, our studies also revealed a significant amount of not only Cldn6 but also Cldn10, Cldn11, and Cldn18 in the cytoplasm of transgenic epidermal cells as well as the activation of a protein-unfolding pathway. These findings demonstrate that the overexpression of a tail truncation mutant of Cldn6 mislocalizes Cldn6 and other Cldn proteins to the cytoplasm and triggers a postnatal increase in proliferation and aberrant differentiation of the epidermis, emphasizing the importance of the Cldn tail domain in membrane targeting and function in vivo.

Amino Acid Sequence↗

PubMed2006

Derivation of epidermal colony-forming progenitors from embryonic stem cell cultures.

In our two-step culturing system, epidermal progenitor cells derived from differentiating embryonic stem cells in vitro are plated on either a basement membrane matrix or in coculture. On plating on basement membrane, differentiation along the epidermal lineage is evident and reproducible, with several markers of epidermal differentiation observed in a pattern reminiscent of their in vivo expression. However, when epidermal progenitor cells are plated in a co-culture environment with mature epidermal cells at low density, colony formation, and differentiation along the epidermal pathway is enhanced with a high frequency of K14-positive cells. Thus, the in vitro co-culture model system described here is useful in dissecting the process of commitment and differentiation of epithelial progenitors that occurs during epidermogenesis as well as for the investigation of the molecular mechanisms underlying these processes.

Animals↗

PubMed2006

Directing epidermal fate selection by a novel co-culture system.

We describe an improved culturing protocol to direct embryonic stem (ES) cell differentiation along the epidermal lineage in vitro. Pluripotent ES cells co-cultured with mitomycin C-treated mature epidermal cells at low density resulted in colonies that undergo differentiation to keratin 14 (K14)-positive cells within 3 d in culture. This new protocol suggests that the commitment and differentiation of undifferentiated ES cells can be efficiently directed by mature epidermal cell-derived factors. In addition, it suggests that this condition may be responsible for the maintenance and expansion of putative epidermal stem cells in vitro. Our advanced culturing regimen allows for the analysis of colony formation from a single cell to determine the factors controlling the differentiation capacities of very early progenitor cells in culture.

Animals↗

PubMed2006

Human embryonic stem cells: isolation, maintenance, and differentiation.

The isolation of pluripotent human embryonic stem (hES) cells having the capacity to differentiate in vitro to numerous cell types generated much excitement and promise in the field of regenerative medicine. However, along with great enthusiasm came hot controversy for stem cell research and researchers alike because available hES cell lines were isolated from "excess" embryos from in vitro fertilization clinics. Despite ethical and political debates, the methods and protocols to study diverse lineages are developing. Furthermore, strategies using specific growth factor combinations, cell-cell and cell-extracellular matrix induction systems are being explored for directed differentiation along a desired lineage. However, there is a great need to characterize the mechanisms that control self-renewal and differentiation and a necessity to improve methodologies and develop new purification protocols for the potential future clinical application of hES cells. After the scientific and political obstacles are overcome, it is anticipated that the hES cell field will make a tremendous difference in conditions, such as burn traumas and diabetic foot ulcers, as well a number of degenerative diseases such as Parkinson's disease, type 1 diabetes, rheumatoid arthritis, and myocardial infarction. In this introductory chapter, we will summarize and review recent progress in the field of hES cell differentiation protocols and discuss some of the current issues surrounding hES cell research.

Cell Culture Techniques↗

PubMed2005

Delayed epidermal permeability barrier formation and hair follicle aberrations in Inv-Cldn6 mice.

Homozygous mice overexpressing Claudin-6 (Cldn6) exhibit a perturbation in the epidermal differentiation program leading to a defective epidermal permeability barrier (EPB) and dehydration induced death ensuing within 48 h of birth [Turksen, K., Troy, T.C., 2002. Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 129, 1775-1784]. Their heterozygous counterparts are also born with an incomplete EPB; however, barrier formation continues after birth and normal hydration levels are achieved by postnatal day 12 allowing survival into adulthood. Heterozygous Inv-Cldn6 mice exhibit a distinct coat phenotype and histological analysis shows mild epidermal hyperkeratosis. Expression of K5 and K14 is aberrant, extending beyond the basal layer into the suprabasal layer where they are not co-localized suggesting that their expression is uncoupled. There is also atypical K17 and patchy K15 expression in the basal layer with no K6 expression in the interfollicular epidermis; together with marked changes in late differentiation markers (e.g. profilaggrin/filaggrin, loricrin, transglutaminase 3) indicating that the normal epidermal differentiation program is modified. The expression compartment of various Cldns is also perturbed although overall protein levels remained comparable. Most notably induction of Cldn5 and Cldn8 was observed in the Inv-Cldn6 epidermis. Heterozygous Inv-Cldn6 animals also exhibit subtle alterations in the differentiation program of the hair follicle including a shorter anagen phase, and altered hair type distribution and length compared to the wild type; the approximately 20% increase in zig-zag hair fibers at the expense of guard hairs and the approximately 30% shorter guard hairs contribute to coat abnormalities in the heterozygous mice. In addition, the transgenic hair follicles exhibit a decreased expression of K15 as well as some hair-specific keratins and express Cldn5 and Cldn18, which are not detectable in the wild type. These data indicate that Cldn6 plays a role in the differentiation processes of the epidermis and hair follicle and supports the notion of a link between Cldn regulation and EPB assembly/maintenance as well as the hair cycle.

Animals↗

PubMed2005

Commitment of embryonic stem cells to an epidermal cell fate and differentiation in vitro.

The epidermis develops from a stem cell population in the surface ectoderm that feeds a single vertical terminal differentiation pathway. To date, however, the limited capacity for the isolation or purification of epidermal stem or precursor cells has hampered studies on early commitment and differentiation events. We have developed a two-step culture scheme in which pluripotent mouse embryonic stem (ES) cells are induced first to a surface ectoderm phenotype and then are positively selected for putative epidermal stem cells. We show that the earliest stages of epidermal development follow an ordered sequence that is similar to that observed in vivo (expression of keratin 8, keratin 19, keratin 17, and keratin 14), suggesting that ES cell-derived surface ectoderm-like cells can be induced to follow the epidermal developmental pathway. At a low frequency, keratin 14-positive early epidermal cells progressed to keratin 1-positive and terminally differentiated cells producing a cornified envelope. This culturing protocol provides an invaluable system in which to study both the mechanisms that direct stem cells along the epidermal pathway as well as those that influence their subsequent epidermal differentiation.

Alkaline Phosphatase↗

PubMed2005

Immunolocalization in the epidermis.

Immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment in the epidermis thereby providing evidence as to their functionality. This chapter therefore describes a dependable method for immunolocalization within the epidermis.

Animals↗

PubMed2005

Epidermal cell analysis by RT-PCR.

Reverse transcription polymerase chain reaction is used for the semiquantitative analysis of epidermal gene expression, particularly when immunolocalization is not feasible because of the lack of antibodies available for novel genes. This chapter is therefore devoted to the delineation of a reliable reverse transcription polymerase chain reaction method to analyze gene expression both in vivo and in vitro.

Animals↗

PubMed2004

Barriers built on claudins.

The fundamental functions of epithelia and endothelia in multicellular organisms are to separate compositionally distinct compartments and regulate the exchange of small solutes and other substances between them. Tight junctions (TJs) between adjacent cells constitute the barrier to the passage of ions and molecules through the paracellular pathway and function as a 'fence' within the plasma membrane to create and maintain apical and basolateral membrane domains. How TJs achieve this is only beginning to be understood. Recently identified components of TJs include the claudins, a family of four-transmembrane-span proteins that are prime candidates for molecules that function in TJ permeability. Their identification and characterization have provided new insight into the diversity of different TJs and heterogeneity of barrier functions in different epithelia and endothelia.

Amino Acid Motifs↗

PubMed2004

Revisiting the bulge.

Two recent papers--one from the group of Elaine Fuchs in Science and the other from the group of George Costarelis in Nature Biotechnology--on the isolation and multipotentiality of adult hair follicle stem cells catapult us ahead in our understanding of epidermal and hair follicle lineages.

Animals↗

PubMed2003

Overexpression of the calcium sensing receptor accelerates epidermal differentiation and permeability barrier formation in vivo.

The calcium sensing receptor (CaSR) has emerged as an important mediator of a wide range of Ca(2+)-dependent physiological responses (Ca(2+) signaling) in various tissues. To explore the role of CaSR in the epidermis, we utilised the keratin 14 promoter to express CaSR cDNA constitutively in the basal cells of the stratified squamous epithelium of transgenic mice. Analysis of the transgenic mice revealed that a sensitized response to CaSR signaling accelerates the epidermal differentiation program with the precocious formation of the epidermal permeability barrier (EPB) during development and an accelerated hair growth at birth. Our observations indicate that overexpression of CaSR in the undifferentiated basal cells leads to changes in the differentiation program of the transgenic epidermis, including the stimulation of keratins 1 and 6 as well as the overexpression of several markers of terminal differentiation such as filaggrin, loricrin and involucrin. Our data suggest that the observed modifications in the differentiation pathway are a consequence of a CaSR-induced enhancement of Ca(2+) signaling involving cross-talk with other signaling pathways (e.g. EGF and Wnt/Ca(2+)). These studies provide new insights into the role of CaSR in epidermal differentiation including EPB development and hair follicle morphogenesis.

Animals↗

PubMed2002

Permeability barrier dysfunction in transgenic mice overexpressing claudin 6.

A defective epidermal permeability barrier (EPB) in premature birth remains a leading cause of neonatal death as a result of its associated complications, which include poor temperature stability, infection by micro-organisms through the skin, and the outflow of water. Despite its importance in survival, the mechanisms involved in the formation and maintenance of the EPB are not well understood. To address the possibility that claudins, a new superfamily of tight junctional molecules, are involved, we engineered transgenic mice with claudin 6 (Cldn6) overexpressed via the involucrin (Inv) promoter. Interestingly, the Inv-Cldn6 transgenic animals die within 2 days of birth, apparently due to the lack of an intact EPB as evidenced by increased water loss and the penetration of X-gal through the skin. Barrier dysfunction was manifested biochemically by the aberrant expression of late epidermal differentiation markers, including K1, filaggrin, loricrin, transglutaminase 3, involucrin, repetin, members of the SPRR family and the transcriptional regulator Klf4. The overall claudin profile of the epidermis was also modified. Our data suggest that repetin and SPRR1A and 2A are downregulated in response to the downregulation of Klf4 in the transgenic animals, which would contribute to decreased protein crossbridging leading to fragile, defective cornified envelopes. These results provide new insights into the role of claudin 6 in epithelial differentiation and EPB formation. In addition, the epidermal phenotype of these transgenic mice, which is very reminiscent of that in pre-term infant skin, suggest that they will be an important and novel model for studies on human premature EPB-related morbidity.

Animals↗