P2X5 and P2X7 receptors in human warts and CIN-612 organotypic raft cultures of human papillomavirus infected keratinocytes
7
Citation
37
Reference
10
Related Paper
Citation Trend
Abstract:
Purinergic receptors, which bind adenosine 5'-triphosphate (ATP), are expressed on human cutaneous keratinocytes and in squamous cell carcinomas. Studies on normal human epidermis and primary keratinocyte cultures have suggested that P2X(5) receptors are likely to be involved in keratinocyte differentiation and P2X(7) receptors are likely to be part of the machinery of end stage terminal differentiation/apoptosis of keratinocytes. P2X(7) receptor agonists can significantly reduce primary keratinocyte cell numbers in culture. Human papillomaviruses are increasingly recognised as important human carcinogens in the development of non-melanoma skin cancers. In our study, immunohistochemical analysis for P2X(5) and P2X(7) receptors was performed on paraffin sections of normal human skin, warts, raft cultures of normal human keratinocytes and raft cultures of CIN 612 cells, a model of keratinocytes infected with human papillomavirus type 31. In warts there was up-regulation of the expression of P2X(5) receptors. A similar pattern was seen in the CIN 612 raft cultures. Both P2X(5) and P2X(7) receptors were found in the nuclei of koilocytes, abnormal keratinocytes characteristic of human papillomavirus infection. P2X(5) and P2X(7) receptors may provide a new focus for therapeutic research into treatments for warts because these receptors can induce cell differentiation and cell death.Keywords:
Human skin
Epidermis (zoology)
Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y 2 receptors by human keratinocytes, the cells which comprise the epidermis. Reverse transcriptase‐polymerase chain reaction (RT–PCR) revealed expression of mRNA for the G‐protein‐coupled, P2Y 2 receptor in primary cultured human keratinocytes. In situ hybridization studies of skin sections revealed that P2Y 2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y 2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. Increases in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPγS were found to stimulate the proliferation of keratinocytes. Using a real‐time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. These findings indicate that P2Y 2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis. British Journal of Pharmacology (1999) 127 , 1680–1686; doi: 10.1038/sj.bjp.0702653
Epidermis (zoology)
P2Y receptor
Bromodeoxyuridine
Homeostasis
Cite
Citations (109)
Summary Tissue homeostasis requires regulation of cell-cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete specific factors transduced by melanocytes into signaling cues to promote their pigmentation and dendrite outgrowth, while melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How epidermal cells integrate these functions remains poorly characterized. Here, we found that caveolae polarize in melanocytes and are particularly abundant at melanocyte-keratinocyte interface. Caveolae in melanocytes are sensitive to ultra-violet radiations and miRNAs released by keratinocytes. Preventing caveolae formation in melanocytes results in increased production of intracellular cAMP and melanin pigments, but decreases cell protrusions, cell-cell contacts, pigment transfer and epidermis pigmentation. Altogether, our data establish that, in melanocytes, caveolae serve as key molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity. This process is crucial to maintain cell-cell contacts and intercellular communication, skin pigmentation and tissue homeostasis.
Melanocyte
Cell Signaling
Epidermis (zoology)
Human skin
Cite
Citations (2)
Abstract Receptors for melanotropin (MSH) were found to be expressed by immortalized primary human epidermal keratinocytes (RHEK‐1). Using 125 I‐βMSH as a probe, the MSH receptors from mouse melanoma cells and human keratinocytes were found to be remarkably similar. In each cell line, there were high and low affinity receptors, with the high affinity classes showing positive cooperativity. Competition of 125 l‐βMSH for binding with non‐radioactive MSH revealed similar profiles. Cross‐linking studies, followed by gel electrophoresis and autoradiography, showed almost identical gel migration patterns. Both cell types expressed internal as well as plasma membrane binding sites. MSH receptors on both cell types were up‐regulated by ultraviolet light and by MSH itself. Although the function of MSH receptors expressed by the immortalized keratinocytes is unknown, the results are consistent with recent reports that proliferation of epidermal keratinocytes is stimulated by MSH and that proopiomelanocortin genes are expresed in the epidermis. These results support a model in which keratinocytes and melanocytes, interacting in an “epidermal‐melanin unit,” each respond to UV light signals with increased MSH receptor activity. © 1993 Wiley‐Liss, Inc.
Epidermis (zoology)
Human skin
Cell surface receptor
Cite
Citations (60)
Purinergic receptors, which bind adenosine 5'-triphosphate (ATP), are expressed on human cutaneous keratinocytes and in squamous cell carcinomas. Studies on normal human epidermis and primary keratinocyte cultures have suggested that P2X(5) receptors are likely to be involved in keratinocyte differentiation and P2X(7) receptors are likely to be part of the machinery of end stage terminal differentiation/apoptosis of keratinocytes. P2X(7) receptor agonists can significantly reduce primary keratinocyte cell numbers in culture. Human papillomaviruses are increasingly recognised as important human carcinogens in the development of non-melanoma skin cancers. In our study, immunohistochemical analysis for P2X(5) and P2X(7) receptors was performed on paraffin sections of normal human skin, warts, raft cultures of normal human keratinocytes and raft cultures of CIN 612 cells, a model of keratinocytes infected with human papillomavirus type 31. In warts there was up-regulation of the expression of P2X(5) receptors. A similar pattern was seen in the CIN 612 raft cultures. Both P2X(5) and P2X(7) receptors were found in the nuclei of koilocytes, abnormal keratinocytes characteristic of human papillomavirus infection. P2X(5) and P2X(7) receptors may provide a new focus for therapeutic research into treatments for warts because these receptors can induce cell differentiation and cell death.
Human skin
Epidermis (zoology)
Cite
Citations (7)
HaCaT
Immortalised cell line
Epidermis (zoology)
Human skin
Cite
Citations (1)
In order to characterize connexin expression and regulation in the epidermis, we have characterized a rat epidermal keratinocyte (REK) cell line that is phenotypically similar to basal keratinocytes in that they have the ability to differentiate into organotypic epidermis consisting of a basal cell layer, 2–3 suprabasal cell layers, and a cornified layer. RT-PCR revealed that REK cells express mRNA for Cx26, Cx31, Cx31.1, Cx37, and Cx43, which mimics the reported connexin profile for rat tissue. In addition, we report the expression of Cx30, Cx30.3, Cx40, and Cx45 in rat keratinocytes, highlighting the complexity of the connexin complement in rat epidermis. Furthermore, 3-dimensional analysis of organotypic skin revealed that Cx26 and Cx43 are exquisitely regulated during the differentiation process. The life-cycle of these connexins including their expression, transport, assembly into gap junctions, internalization, and degradation are elegantly depicted in organotypic epidermis as keratinocytes proceed from differentiation to programmed cell death.
Epidermis (zoology)
Cite
Citations (31)
Programmed cell death of epidermal keratinocytes occurs in two forms – classical apoptosis after irreparable cellular injuries and during terminal keratinocyte differentiation – leading to stratum corneum formation. Whereas the former is indistinguishable from apoptosis in other tissues and leads to cellular disintegration, the latter results in establishment of ‘indestructible’ cellular ghosts. Although there are several similarities between these two forms of programmed cell death such as the loss of the nucleus and cellular organelles, the persistence of dead keratinocytes as corneocytes represents a major difference. As to the molecular events which drive both processes, many known factors of the pro- and anti-apoptotic machinery are expressed by keratinocytes and appear to become active after UV irradiation or other damaging insults but not in the last phase of keratinocyte differentiation. By contrast, distinct members of the pro-apoptotic enzyme families such as caspases and DNases are expressed almost exclusively in epidermal keratinocytes. Most notably, caspase-14 and DNAse1L2 are upregulated and activated in a differentiation-associated manner. This suggests that certain elements of the molecular apoptotic machinery have adapted to specific roles in terminal differentiation-associated cell death in the epidermis. Allombert-Blaise C, Tamiji S, Mortier L, et al. Terminal differentiation of human epidermal keratinocytes involves mitochondria- and caspase-dependent cell death pathway. Cell Death Differ 2003: 10: 850-852. This article evidenced that the differentiation process in epidermal keratinocytes uses mitochondrial-dependent death machinery and suggested that differentiation is regulated by a balance of prepression and activation between proliferation and apoptosis. Candi E, Schmidt R, Melino G. The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol 2005: 6: 328-340. This article is an excellent overview on the insights into the molecular mechanisms and the physiological endpoints of cornification and the current understanding of the pathological defects of this form of programmed cell death. Chang CH, Yu M, Wu P, et al. Sculpting skin appendages out of epidermal layers via temporally and spatially regulated apoptotic events. J Invest Dermatol 2004: 122 (6): 1348-1355. This article examines apoptosis in the morphogenesis of skin appendages and shows how adding and removing cell masses in temporally and spatially specific ways are coordinated to sculpt skin appendages from epidermal layers. Chaturvedi V, Qin JZ, Denning MF, Choubey D, Diaz MO, Nickoloff BJ. Apoptosis in proliferating, senescent, and immortalized keratinocytes. J Biol Chem 1999: 274: 23358-23367. The authors demonstrate that growth arrest, confluency, and senescence of keratinocytes are associated with resistance to classical apoptosis and that differentiation of keratinocytes is no prerequisite for conventional apoptosis induction. Eckhart L, Declercq W, Ban J, et al. Terminal differentiation of human keratinocytes and stratum corneum formation is associated with caspase-14 activation. J Invest Dermatol 2000: 115: 1148-1151. Demonstration that expression and activation of an epidermis-specific caspase correlates with terminal keratinocyte differentiation in vitro. Gandarillas A, Goldsmith LA, Gschmeissner S, Leigh IM, Watt FM. Evidence that apoptosis and terminal differentiation of epidermal keratinocytes are distinct processes. Exp Dermatol 1999: 8: 71-79. Comparison of normal epidermis and epidermis from skin diseases with disturbed differentiation with no evidence of increased apoptosis in the latter despite a higher proportion of TUNEL-positive cells (suggestive that TUNEL is not a specific marker of apoptosis in keratinocytes). Together with in vitro data conclusion that in vivo and in culture keratinocyte terminal differentiation and apoptosis are distinct cellular events subject to different stimuli. Kuechle MK, Presland RB, Lewis SP, Fleckman P, Dale BA. Inducible expression of filaggrin increases keratinocyte susceptibility to apoptotic cell death. Cell Death Differ 2000: 7: 566-573 The authors generated keratinocyte cell lines expressing mature human filaggrin under an inducible promoter system and found that these cell lines exhibited increased sensitivity to multiple apoptotic stimuli. They conclude that filaggrin, which is expressed at a level of the epidermis where keratinocytes are in transition between the nucleated granular and the anucleate cornified layers, aids in the terminal differentiation process by facilitating apoptotic machinery. Pena JC, Fuchs E, Thompson CB. Bcl-x expression influences keratinocyte cell survival but not terminal differentiation. Cell Growth Differ 1997: 8: 619-629. Overexpression of bcl-xL or bcl-xS under the control of the K14 promoter did not compromise the maturation process and cellularity of the epidermis but dramatically increase resistance (bcl-xL) or sensitivity (bcl-xS) to irradiation. Their findings demonstrate that the terminal differentiation program of keratinocyte is not altered by the overexpression of anti-apoptotic proteins. Takahashi H, Aoki N, Nakamura S, Asano K, Ishida-Yamamoto A, Iizuka H. Cornified cell envelope formation is distinct from apoptosis in epidermal keratinocytes. J Dermatol Sci 2000: 23: 161-169. The authors analyzed the effects of the calcium ionophore A23187 and UV on keratinocytes and demonstrated that although both treatments lead to the cell death of keratinocyte, they followed two distinct pathways (i.e. conventional apoptosis and formation of cornified envelope respectively) and concluded that CE formation is distinct from apoptosis in epidermal keratinocytes. Weil M, Raff MC, Braga VM. Caspase activation in the terminal differentiation of human epidermal keratinocytes. Curr Biol 1999: 9: 361-364. The authors report that pro-apoptotic caspases are activated during normal human keratinocyte differentiation and that this activation is apparently required for the normal loss of the nucleus. Wrone-Smith T, Johnson T, Nelson B, et al. Discordant expression of Bcl-x and Bcl-2 by keratinocytes in vitro and psoriatic keratinocytes in vivo. Am J Pathol 1995: 146: 1079-1088. The authors analyzed the expression of pro- and anti-apoptotic regulators in keratinocytes in vitro and in vivo and suggested that the overexpression of Bcl-x in psoriasis may contribute to the longevity of keratinocytes by blocking the normal apoptotic process involved in the terminal differentiation.
Epidermis (zoology)
Corneocyte
Cite
Citations (2)
We previously demonstrated that calcium propagation plays a crucial role in epidermal homeostasis when the epidermis was exposed to a dry environment. In the present study, we first demonstrated the intracellular calcium oscillation in cultured human skin keratinocytes. On partial exposure of cultured human keratinocytes to air, a transient increase of intracellular calcium concentration appeared, followed by a wave-like increase in the unexposed keratinocytes, showing oscillations with a frequency that varied from cell to cell. There appeared to be no correlation between the oscillation frequencies in adjacent cells. The increase of calcium concentration did not appear when calcium was removed from the medium or when suramin, a purinergic receptor antagonist, was added. The ATP concentration also increased immediately after keratinocytes were exposed to air. We hypothesize that ATP is secreted from keratinocytes on exposure to air, and induces an increase of intracellular calcium concentration.
Calcium in biology
Epidermis (zoology)
Human skin
Cite
Citations (38)
The strong societal urge to reduce the use of experimental animals, and the biological differences between rodent and human skin, have led to the development of alternative models for healthy and diseased human skin. However, the limited availability of primary keratinocytes to generate such models hampers large-scale implementation of skin models in biomedical, toxicological, and pharmaceutical research. Immortalized cell lines may overcome these issues, however, few immortalized human keratinocyte cell lines are available and most do not form a fully stratified epithelium. In this study we compared two immortalized keratinocyte cell lines (N/TERT1, N/TERT2G) to human primary keratinocytes based on epidermal differentiation, response to inflammatory mediators, and the development of normal and inflammatory human epidermal equivalents (HEEs). Stratum corneum permeability, epidermal morphology, and expression of epidermal differentiation and host defence genes and proteins in N/TERT-HEE cultures was similar to that of primary human keratinocytes. We successfully generated N/TERT-HEEs with psoriasis or atopic dermatitis features and validated these models for drug-screening purposes. We conclude that the N/TERT keratinocyte cell lines are useful substitutes for primary human keratinocytes thereby providing a biologically relevant, unlimited cell source for in vitro studies on epidermal biology, inflammatory skin disease pathogenesis and therapeutics.
Immortalised cell line
Human skin
Epidermis (zoology)
Cell type
Cite
Citations (163)
Purinergic receptors, which bind adenosine 5'-triphosphate (ATP), are expressed on human cutaneous keratinocytes and in squamous cell carcinomas. Studies on normal human epidermis and primary keratinocyte cultures have suggested that P2X(5) receptors are likely to be involved in keratinocyte differentiation and P2X(7) receptors are likely to be part of the machinery of end stage terminal differentiation/apoptosis of keratinocytes. P2X(7) receptor agonists can significantly reduce primary keratinocyte cell numbers in culture. Human papillomaviruses are increasingly recognised as important human carcinogens in the development of non-melanoma skin cancers. In our study, immunohistochemical analysis for P2X(5) and P2X(7) receptors was performed on paraffin sections of normal human skin, warts, raft cultures of normal human keratinocytes and raft cultures of CIN 612 cells, a model of keratinocytes infected with human papillomavirus type 31. In warts there was up-regulation of the expression of P2X(5) receptors. A similar pattern was seen in the CIN 612 raft cultures. Both P2X(5) and P2X(7) receptors were found in the nuclei of koilocytes, abnormal keratinocytes characteristic of human papillomavirus infection. P2X(5) and P2X(7) receptors may provide a new focus for therapeutic research into treatments for warts because these receptors can induce cell differentiation and cell death.
Human skin
Cite
Citations (7)