Delphinidin,a dietary antioxidant,induces human epidermal keratinocyte differentiation but not apoptosis: studies in submerged and three‐dimensional epidermal equivalent models

Delphinidin (Del), [3,5,7,3'‐,4'‐,5'‐hexahydroxyflavylium], an anthocyanidin and a potent antioxidant abundantly found in pigmented fruits and vegetables exhibits proapoptotic effects in many cancer cells. Here, we determined the effect of Del on growth, apoptosis and differentiation of normal human epidermal keratinocytes (NHEKs) in vitro in submerged cultures and examined its effects in a three‐dimensional (3D) epidermal equivalent (EE) model that permits complete differentiation reminiscent of in vivo skin. Treatment of NHEKs with Del (10–40 μm ; 24–48 h) significantly enhanced keratinocyte differentiation. In Del‐treated cells, there was marked increase in human involucrin (hINV) promoter activity with simultaneous increase in the mRNA and protein expressions of involucrin and other epidermal differentiation markers including procaspase‐14 and transglutaminase‐1 (TGM1), but without any effect on TGM2. Del treatment of NHEKs was associated with minimal decrease in cell viability, which was not associated with apoptosis as evident by lack of modulation of caspases, apoptosis‐related proteins including Bcl‐2 family of proteins and poly(ADP‐ribose) polymerase cleavage. To establish the in vivo relevance of our observations in submerged cultures, we then validated these effects in a 3D EE model, where Del was found to significantly enhance cornification and increase the protein expression of cornification markers including caspase‐14 and keratin 1. For the first time, we show that Del induces epidermal differentiation using an experimental system that closely mimics in vivo human skin. These observations suggest that Del could be a useful agent for dermatoses associated with epidermal barrier defects including aberrant keratinization, hyperproliferation or inflammation observed in skin diseases like psoriasis and ichthyoses.     

Growth and Differentiation Properties of Normal and Transformed Human Keratinocytes in Organotypic Culture

The growth and differentiation of human normal keratinocytes and their transformed counterparts were examined in organotypic cultures in which the keratinocytes were grown at the air-liquid interface on top of contracted collagen gel containing fibroblasts. We developed a modified culture procedure including the use of a mixed medium for keratinocytes and fibroblasts. Normal keratinocytes formed a three-dimensional structure of epithelium that closely resembled the epidermis in vivo , consisting of basal, spinous, granular and cornified layers. Cells synthesizing DNA were located in the lowest basal layer facing the collagen gel. Expressions of proteins involved in epidermal differentiation were examined by immunohistochemical staining and compared with those in skin in vivo . In the organotypic culture, transglutaminase, involucrin and filaggrin were expressed, as in the epidermis in vitro , most prominently in the granular layer. Type IV collagen, a component of basement membrane, was expressed at the interface between the keratinocyte sheet and the contracted collagen gel. Keratinocytes transformed by simian virus 40 or human papilloma virus (HPV) exhibited a highly disorganized pattern of squamous differentiation. In particular, HPV-transformed cells invaded the collagen gel. Organotypic culture is unique in that regulatory mechanisms of growth and differentiation of keratinocytes can be investigated under conditions mimicking those in vivo .     

Initiation and characterization of keratinocyte cultures from biopsies of normal human conjunctiva

The purpose of the present study was to establish and characterize serum-free epithelial cultures of normal human conjunctiva using fresh biopsy tissue. To this end, small pieces of normal conjunctiva were biopsied from patients undergoing routine cataract surgery. Fragments of the tissue were placed in explant culture in medium containing fetal bovine serum for approximately 1 week to promote epithelial cell outgrowth. Cultures were then passaged multiple times into serum-free medium. Cultures generated in this way were at least 95% keratinocytes and exhibited a typical epithelial morphology, which was dependent on the extracellular Ca(2+)concentration. Immunocytochemical staining revealed that E-cadherin, P-cadherin, and involucrin were present in the cultures, with distributions consistent with their in vivo localization patterns. Distribution of keratins 19, 3, and 4 in conjunctival epithelial cultures were also consistent with in vivo patterns and distinctly different from patterns observed in epithelial cultures similarly generated from cornea and foreskin. Hence, conjunctival keratinocyte cultures retain some tissue-specific markers and do not revert to a generic, culture phenotype.     

Purinergic receptor expression in the regeneration epidermis in a rat model of normal and delayed wound healing

This study investigated changes in the protein expression of purinergic receptors in the regenerating rat epidermis during normal wound healing, in denervated wounds, and in denervated wounds treated with nerve growth factor (NGF), where wound healing rates are normalized. Excisional wounds were placed within denervated, pedicled, oblique, groin skin flaps, and in the contralateral abdomen to act as a control site. Six rats had NGF-treated wounds and six had untreated wounds. Tissue was harvested at day four after wounding. The re-epithelializing wound edges were analyzed immunohistochemically for P2X(5), P2X(7), P2Y(1) and P2Y(2) receptors, and immunostaining of keratinocytes was quantified using optical densitometry. In normal rat epidermis, P2Y(1) and P2Y(2) receptors were found in the basal layer where keratinocytes proliferate; P2X(5) receptors were associated with proliferating and differentiating epidermal keratinocytes in basal and suprabasal layers; P2X(7) receptors were associated with terminally differentiated keratinocytes in the stratum corneum. In the regenerating epidermis of denervated wounds, P2Y(1) receptor protein expression was significantly increased in keratinocytes (P<0.001) but P2Y(1) receptors (P<0.001) compared with untreated denervated wounds. In innervated wounds, NGF treatment enhanced expression in keratinocytes. P2X(5) (P>0.001) and P2Y(1) receptor protein (P<0.001) expression in keratinocytes. P2X(7) receptors were absent in all experimental wound healing preparations. P2X(5), P2X(7), P2Y(1) and P2Y(2) receptor protein expression in the regenerating epidermis was altered both during wound healing and also by NGF treatment. Possible roles for purinergic signalling and its relation to NGF in wound healing are discussed.     

结缔组织生长因子在口腔黏膜下纤维性变组织中的角质形成细胞中的表达

目的探讨结缔组织生长因子(CTGF)在口腔黏膜下纤维性变(OSF)发病机理中的作用.方法应用原位杂交法对34例OSF、10例正常口腔黏膜组织角质形成细胞(KC)中CTGF mRNA进行检测.结果24例(70.6%)OSF组织KC中有CTGF mRNA表达,早期OSF组织KC中CTGF mRNA阳性表达明显高于中晚期OSF(P＜0.05),10例正常口腔黏膜组织KC中CTGF mRNA表达呈阴性.结论OSF组织KC可合成分泌CTGF,在OSF发病机理中CTGF可能起重要作用,CTGF可能作为一中间介质参与了OSF的发病过程.     

Defective expression of a desmosome-related keratinocyte membrane antigen in squamous cell carcinoma

Intercellular junctions of keratinocytes are essential for coordination of the epithelial tissue function. Neoplastic transformation is often accompanied by changes in the glycolipid and glycoprotein composition of the cell membranes, although no evident structural modifications can be observed. We have investigated one aspect of the keratinocyte cell-cell contact in squamous cell carcinoma using KM48 monoclonal antibody as a differentiation-related marker for desmosomes. Quantitative analysis of the immunogold labelling revealed that the tumor cells lose the KM48 antigen during their neoplastic transformation. The process could also be observed, although to a lesser degree, in clinically and ultrastructurally normal marginal epidermis. The desmosome frequency in the tumor tissue also decreased, but not as radically as did the KM48 expression. Decreased expression of the desmosome-related membrane antigen suggests qualitative and perhaps functional modifications of the junction in squamous cell carcinoma.     

A genetic mechanism for cecal atresia: the role of the Fgf10 signaling pathway

BACKGROUND: Intestinal atresia represents a significant surgically correctable cause of intestinal obstruction in neonates. Intestinal development proceeds as a tube-like structure with differentiation along its axis. As the intestine differentiates, the cecum develops at the transition from small to large intestine. Fgf10 is known to serve a key role in budding morphogenesis; however, little is known about its role in the development of this transitional structure. Here we evaluate the effect of Fgf10/Fgfr2b invalidation on the developing cecum. MATERIALS AND METHODS: Wild-type C57Bl/6, Fgf10(-/-), and Fgfr2b(-/-) embryos harvested from timed pregnant mothers were analyzed for cecal phenotype, Fgf10 expression, and differentiation of smooth muscle actin. RESULTS: Wt cecal development is first evident at E11.5. FGF10 is discreetly expressed in the area of the developing cecum at early stages of development. One hundred percent of Fgf10(-/-) and Fgfr2b(-/-) mutant embryos demonstrate cecal atresia with absence of epithelial and muscular layers. The development of neighboring anatomical structures such as the ileocecal valve is not affected by Fgf10/Fgfr2b invalidation. CONCLUSIONS: FGF10 expression is localized to the cecum early in the normal development of the cecum. Fgf10(-/-) and Fgfr2b(-/-) mutant embryos demonstrate cecal atresia with complete penetrance. Epithelial and muscular layers of the cecum are not present in the atretic cecum. The Fgf10(-/-) and Fgfr2b(-/-) mutants represent a genetically reproducible animal model of autosomal recessive intestinal atresia.     

Mutation frequencies in murine keratinocytes as a function of carcinogenic status

A link between genetic abnormalities and carcinogenesis is well established. It follows that a correlation exists between mutation frequency and malignant progression. We have determined the spontaneous and DNA damage-induced mutation frequencies for a series of cell lines derived from SENCAR mouse keratinocytes at various stages of malignant progression. Nontumorigenic mouse keratinocytes (3PC), papillomas (MT1/2), squamous-cell carcinomas (CH72), and spindle-cell carcinomas (CH72T4) were transfected with damaged or undamaged shuttle vectors containing a supF mutation reporter gene. The plasmid mutation frequencies were determined by blue/white screening. The spontaneous plasmid mutation frequency of the squamous-cell carcinoma line was slightly higher than the mutation frequencies of the other cell lines tested. The DNA damage induced by triplex-directed psoralen crosslinks increased the mutation frequencies sixfold to eighteenfold in all cell lines tested, with no significant differences among the cell lines. Sequence analyses revealed that the spindle-cell carcinoma line had a different spontaneous mutation spectrum from the other cell lines. DNA damage-induced mutations were predominantly point mutations at the triplex-duplex junction in all of the cell lines tested, as expected. These data suggested that a strong mutator phenotype was not required for progression to an advanced malignant phenotype in our model system.