Cartilage-specific overexpression of ERRγ results in Chondrodysplasia and reduced chondrocyte proliferation.

While the role of estrogen receptor-related receptor alpha (ERRα) in chondrogenesis has been investigated, the involvement of ERR gamma (ERRγ) has not been determined. To assess the effect of increased ERRγ activity on cartilage development in vivo, we generated two transgenic (Tg) lines overexpressing ERRγ2 via a chondrocyte-specific promoter; the two lines exhibited ∼3 and ∼5 fold increased ERRγ2 protein expression respectively in E14.5 Tg versus wild type (WT) limbs. On postnatal day seven (P7), we observed a 4-10% reduction in the size of the craniofacial, axial and appendicular skeletons in Tg versus WT mice. The reduction in bone length was already present at birth and did not appear to involve bones that are derived via intramembranous bone formation as the bones of the calvaria, clavicle, and the mandible developed normally. Histological analysis of P7 growth plates revealed a reduction in the length of the Tg versus WT growth plate, the majority of which was attributable to a reduced proliferative zone. The reduced proliferative zone paralleled a decrease in the number of Ki67-positive proliferating cells, with no significant change in apoptosis, and was accompanied by large cell-free swaths of cartilage matrix, which extended through multiple zones of the growth plate. Using a bioinformatics approach, we identified known chondrogenesis-associated genes with at least one predicted ERR binding site in their proximal promoters, as well as cell cycle regulators known to be regulated by ERRγ. Of the genes identified, Col2al, Agg, Pth1r, and Cdkn1b (p27) were significantly upregulated, suggesting that ERRγ2 negatively regulates chondrocyte proliferation and positively regulates matrix synthesis to coordinate growth plate height and organization.

Junctions gone bad: claudins and loss of the barrier in cancer.

The tight junctions (TJs) of epithelia are responsible for regulating the "fence and gate" function of polarized epithelial cells. It is now well-established that dysregulation of these functions contributes to initiation and progression of cancer. Recently, it has become clear that the Claudins, members of a large family of 27 closely related transmembrane proteins, play a crucial role in formation, integrity and function of TJs, the epithelial permeability barrier and epithelial polarization. A growing body of data indicates that Claudin expression is altered in numerous epithelial cancers in a stage- and tumor-specific manner. While a single universal mechanism is still lacking, accumulating evidence supports a role for epigenetic regulation of Claudin expression in tumorgenesis, with concomitant alterations in barrier function. We review here new insights and challenges in understanding Claudin function in normal physiology and cancer.

Re-assessing K15 as an epidermal stem cell marker.

The intermediate filament keratin 15 (K15) is present in variable amounts in various stratified epithelia, but has also been reported to be a stem cell marker in the hair follicle. Using peptide specific antibodies, we evaluated the temporal and spatial distribution pattern of K15 expression/localization during normal epidermal development and initiation of hair follicle formation, and in the injured mature epidermis (e.g., during acute injury and repair and in tumorigenesis). During development, K15 expression is first localized to a subset of epidermal basal cells and the overlying periderm at E12.5, but its expression is seen throughout the basal layer by E15.5 and beyond. In hair follicle morphogenesis, initial peg formation occurs in a K15-null area at E14.5 and as peg elongation proceeds through to the mature hair follicle, K15 expression follows the leading edge with positive cells restricted to the outer root sheath. In an epidermal injury model, K15 is first up-regulated and associated with both the basal and suprabasal layers of the interfollicular epidermis then expression becomes sporadic and down-regulated before a basal layer-specific association is re-established in the repaired epidermis. During tumorigenesis, K15 is first mis-expressed, and is ultimately down-regulated. Our data suggest that K15 protein expression may reflect not only expression in a stem or progenitor cell subpopulation, but also reflects the activity and responsiveness of basal-like cells to loss of homeostasis of the epidermal differentiation program. Thus, the data suggest caution in using K15 alone to delineate epidermal stem cells, and underscore the need for further investigation of K15 and other markers in epidermal cell subpopulations.

Insights into the role of the calcium sensing receptor in epidermal differentiation in vivo.

While the important role of calcium (Ca(++)) signaling is fundamental in epidermal cell physiology, a detailed knowledge of precisely how epidermal cells respond to Ca(++) levels is not clear. Using peptide-specific antibodies that we generated, we set out to evaluate the temporal and spatial distribution pattern of the Ca(++)-sensing receptor (CaSR) during epidermogenesis and to assess its involvement in the mature epidermis (e.g., in acute injury and tumorigenesis). Our data indicate a developmentally regulated expression of CaSR: up-regulation occurs in specific epidermal cells and cell layers in normal development or in response to injury when epidermal cells are induced to undergo commitment and early differentiation events, and down-regulation occurs in terminal differentiation stages. These results provide a new perspective on the role of the CaSR in these processes and describe a novel tool for evaluating Ca(++)-mediated epidermal differentiation.

Changes in the distribution pattern of Claudin tight junction proteins during the progression of mouse skin tumorigenesis.

BACKGROUND: Despite the fact that morphological and physiological observations suggest that the tight junction (TJ)-based permeability barrier is modified/disrupted in tumorigenesis, the role of members of the Claudin (Cldn) family of TJ proteins is not well-understood. Using a well-established two-stage chemical carcinogenesis model, we investigated the temporal and spatial changes in expression of those Cldns that we have previously demonstrated to be important in epidermal differentiation and the formation of the epidermal permeability barrier, i.e., Cldn1, Cldn6, Cldn11, Cldn12 and Cldn18. METHODS: The lower dorsal backskin of mice was treated topically with 7,12-dimethylbenz(a)anthracene (DMBA; 0.25 mg/ml in acetone) and following a 10-day incubation period, 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 25 microg/ml in acetone) was applied three times a week to the same area. Backskin samples were dissected 2, 4, 6, 8 and 12 weeks after the initiation of the experimental protocol and immunohistochemistry was performed on sections using antibodies against the following: Cldn1, Cldn6, Cldn11, Cldn12, Cldn18, Ki67 and CD3. RESULTS: Our data indicate that along with the changes in epidermal cell morphology and differentiation that occur during tumor formation, there is a dramatic change in Cldn distribution consistent with cell polarity and barrier selectivity changes. Specifically, in the early stages of DMBA/TPA treatment, the suprabasal-specific Cldns occupy an expanded zone of expression corresponding to an increased number of suprabasal epidermal cell layers. As tumorigenesis progressed, the number of suprabasal epidermal layers positive for Cldn6, Cldn11, Cldn12 and Cldn18 was reduced, especially in the lower strata of the expanded suprabasal zone. In addition, a variably reduced cell membrane association of those differentiation-specific Cldns was observed, especially within the infiltrating epidermal structures. In contrast, Cldn1 (which is normally expressed in all the living layers of the epidermis) remained restricted to the cell membrane throughout the tumorigenesis protocol. However commencing 2 weeks after treatment there was a marked decrease in the number of Cldn1-positive basal cells, and the zone of Cldn1-null epidermal cells was expanded up into the lower stratified epidermis throughout the progression of DMBA/TPA treatment. In addition, there was no Cldn1 localization in the infiltrating epidermal structures of the tumorigenic epidermis. CONCLUSION: This is the first demonstration of the changes in Cldn expression in the progression of DMBA/TPA-induced skin tumors; however further investigation into the molecular mechanisms regulating the observed changes in barrier selectivity during tumorigenesis is required.

The targeted overexpression of a Claudin mutant in the epidermis of transgenic mice elicits striking epidermal and hair follicle abnormalities.

Skin is one of the largest organs of the body, and is formed during development through a highly orchestrated process involving mesenchymal-epithelial interactions, cell commitment, and terminal differentiation. It protects against microorganism invasion and UV irradiation, inhibits water loss, regulates body temperature, and is an important part of the immune system. Using transgenic mouse technology, we have demonstrated that Claudin (Cldn)-containing tight junctions (TJs) are intricately involved in cell signaling during epidermal differentiation and that an epidermal suprabasal overexpression of Cldn6 results in a perturbed epidermal terminal differentiation program with distinct phenotypic abnormalities. To delineate the role of the Cldn cytoplasmic tail domain in epidermal differentiation, we engineered transgenic mice targeting the overexpression of a Cldn6 cytoplasmic tail-truncation mutant in the epidermis. Transgenic mice were characterized by a lethal barrier dysfunction in addition to the existence of hyperproliferative squamous invaginations/cysts replacing hair follicles. Immunohistochemical analysis revealed an epidermal cytoplasmic accumulation of Cldn6, Cldn11, Cldn12, and Cldn18, downregulation of Cldn1 and aberrant expression of various classical markers of epidermal differentiation; namely the basal keratins as well as K1, involucrin, loricrin, and filaggrin. Collectively these studies suggest an important role for Cldns in epidermal/hair follicle differentiation programs likely involving cross talk to signaling pathways (e.g., Notch) directing cell fate selection and differentiation.

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.