Primary Epidermal Keratinocytes Research Articles

Characterization of liver X receptor expression and function in human skin and the pilosebaceous unit

The nuclear receptors liver X receptor alpha (LXRalpha) and liver X-receptor beta (LXRbeta) have a well documented role in cholesterol homeostasis and lipid metabolism within tissues and cells including the liver, small intestine and macrophages. In keratinocytes, LXRs have been shown to up-regulate differentiation in vitro via increased transcription of proteins of the AP1 complex and to down-regulate proliferation in vivo. In this study, we provide a detailed description of the location and possible role of LXRs within human skin and its associated glands and appendages. Using RT-PCR, Western blotting and immunohistochemistry, we have demonstrated expression of LXRalpha and LXRbeta mRNA and proteins in whole human skin as well as within a range of primary and immortalized human cell lines derived from human skin, hair follicle and sebaceous glands. Furthermore, we have shown that synthetic LXR specific agonists GW683965 and TO901317 significantly inhibit cell proliferation in primary epidermal keratinocytes, immortalized N/TERT keratinocytes and the immortalized SZ95 sebocyte line, and significantly increase lipogenesis in SZ95 sebocytes. In addition, we showed that the synthetic agonist TO901317 significantly reduced hair growth, in vitro.

In vitro cytotoxicology model of oligo-chitosan and n, o-carboxymethyl chitosan using primary normal human epidermal keratinocyte cultures.

Chitosan (beta-1, 4-D-glucosamine) is a deacetylated form of chitin with excellent biological properties in wound management. The natural properties of chitosan have the physical and chemical limitations to be widely used in biomedical fields. The improvement of the physical and chemical properties of chitosan with some additional chemicals will alter its biocompatibility. Therefore, the biological attribute of the modified chitosan must be evaluated. In this study, the cytotoxicity of oligo-chitosan (OC) and N, O- carboxymethyl-chitosan (NO-CMC) derivatives (O-C 1%, O-C 5%, NO-CMC 1% and NO-CMC 5%) was evaluated using primary normal human epidermal keratinocyte (pNHEK) cultures as an in vitro toxicology model at standardized cell passages (fourth passages). 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl tetrazolium bromide (MTT) was used as a cell viability assay. The O-C 1% is one of the most compatible chitosan derivatives because it steadily sustained >70% of viable cells until 72 hr post-treatment. This was followed by O-C 5%, NO-CMC 5% and NO-CMC 1%. Therefore, oligo-chitosan had the ideal properties of a biocompatible material compared to N, O- carboxymethyl-chitosan in this study.

Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1

To address the functions of Rac1 in keratinocytes of the basal epidermal layer and in the outer root sheath of hair follicles, we generated transgenic mice expressing a dominant inhibitory mutant of Rac, N17Rac1, under the control of the keratin 14 promoter. These mice do not exhibit an overt skin phenotype but show protracted skin wound re-epithelialization. Investigation into the underlying mechanisms revealed that in vivo both proliferation of wound-edge keratinocytes and centripetal migration of the neo-epidermis were impaired. Similar results were obtained in mice with an epidermis-specific deletion of Rac1. Primary epidermal keratinocytes that expressed the N17Rac1 transgene were less proliferative than control cells and showed reduced ERK1/2 phosphorylation upon growth factor stimulation. Adhesion, spreading, random migration and closure of scratch wounds in vitro were significantly inhibited on collagen I and, to a lesser extent, on fibronectin. Stroboscopic analysis of cell dynamics (SACED) of N17Rac1 transgenic and control keratinocytes identified decreased lamella-protrusion persistence in connection with increased ruffle frequency as a probable mechanism for the observed impairment of keratinocyte adhesion and migration. We conclude that Rac1 is functionally required for normal epidermal wound healing and, in this context, exerts a dual function - namely the regulation of keratinocyte proliferation and migration.

Expression of Human Beta Defensin 4 in Genetically Modified Keratinocytes Enhances Antimicrobial Activity

Defensins are cationic peptides of the innate host defense system with antimicrobial activity against many of the microorganisms commonly found in burn units. Beta defensins are variably expressed in the epithelia of skin and other organs. Human beta defensin 4 reportedly has antimicrobial activity against Pseudomonas aeruginosa and is not normally expressed in intact skin. Genetic modification was used to ectopically express human beta defensin 4 in cultured primary epidermal keratinocytes. Keratinocytes expressing human beta defensin 4 showed significantly elevated antimicrobial activity against clinically-isolated P. aeruginosa compared with controls. These results suggest that genetic modification of keratinocytes can increase their resistance to microbial contamination. Bioengineered skin replacements containing human beta defensin 4-modified keratinocytes may be useful for transplantation to contaminated burn wounds.

Streptolysin O enhances keratinocyte migration and proliferation and promotes skin organ culture wound healing in vitro

ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is currently being investigated as a treatment for collagen-related disorders such as scleroderma and fibrosis. Furthermore, ML-05 may be effective in promoting wound healing and alleviating the formation of hypertrophic scars and keloids. To investigate the effects of ML-05 on wound-healing processes, in vitro wound-healing scratch assays (using human primary epidermal keratinocytes and dermal fibroblasts) and a human skin organ culture wound model were utilized. ML-05 markedly enhanced keratinocyte migration and proliferation in wound scratch assays. ML-05 did not affect either proliferation or migration of dermal fibroblasts, indicating that ML-05's effects on cell migration/proliferation may be keratinocyte-specific. ML-05 was tested in a dose-dependent manner in a skin organ culture wound model using two different application methods: Through the culture media (dermal exposure) or direct topical treatment of the wound surface. ML-05 was found to accelerate wound healing as measured by reepithelialization, particularly after topical application. Therefore, ML-05 may have potential as a wound-healing agent that promotes reepithelialization through stimulation of keratinocyte migration and proliferation.

Role of HPV E6 proteins in preventing UVB-induced release of pro-apoptotic factors from the mitochondria

Apoptotic elimination of UV-damaged cells from the epidermis is an important step in preventing both the emergence and expansion of cells with carcinogenic potential. A pivotal event in apoptosis is the release of apoptogenic factors from the mitochondria, although the mechanisms by which the different proteins are released are not fully understood. Here we demonstrate that UV radiation induced the mitochondrial to nuclear translocation of apoptosis inducing factor (AIF) in normal skin. The human papillomavirus (HPV) E6 protein prevented release of AIF and other apoptotic factors such as cytochrome c and Omi from mitochondria of UV-damaged primary epidermal keratinocytes and preserved mitochondrial integrity. shRNA silencing of Bak, a target for E6-mediated proteolysis, demonstrated the requirement of Bak for UV-induced AIF release and mitochondrial fragmentation. Furthermore, screening non-melanoma skin cancer biopsies revealed an inverse correlation between HPV status and AIF nuclear translocation. Our results indicate that the E6 activity towards Bak is a key factor that promotes survival of HPV-infected cells that facilitates tumor development.

Bone morphogenetic protein signaling regulates the size of hair follicles and modulates the expression of cell cycle-associated genes

Bone morphogenetic protein (BMP) signaling is involved in the regulation of a large variety of developmental programs, including those controlling organ sizes. Here, we show that transgenic (TG) mice overexpressing the BMP antagonist noggin (promoter, K5) are characterized by a marked increase in size of anagen hair follicles (HFs) and by the replacement of zig-zag and auchen hairs by awl-like hairs, compared with the age-matched WT controls. Markedly enlarged anagen HFs of TG mice show increased proliferation in the matrix and an increased number of hair cortex and medulla cells compared with WT HFs. Microarray and real-time PCR analyses of the laser-captured hair matrix cells show a strong decrease in expression of Cdk inhibitor p27(Kip1) and increased expression of selected cyclins in TG vs. WT mice. Similar to TG mice, p27(Kip1) knockout mice also show an increased size of anagen HFs associated with increased cell proliferation in the hair bulb. Primary epidermal keratinocytes (KC) from TG mice exhibit significantly increased proliferation and decreased p27(Kip1) expression, compared with WT KC. Alternatively, activation of BMP signaling in HaCaT KC induces growth arrest, stimulates p27(Kip1) expression, and positively regulates p27(Kip1) promoter activity, thus further supporting a role of p27(Kip1) in mediating the effects of BMP signaling on HF size. These data suggest that BMP signaling plays an important role in regulating cell proliferation and controls the size of anagen HFs by modulating the expression of cell-cycle-associated genes in hair matrix KC.

BMP2 and BMP6 control p57 Kip2 expression and cell growth arrest/terminal differentiation in normal primary human epidermal keratinocytes

Functional studies of the canonical Bone Morphogenetic Protein (BMP) signalling pathway in human epidermal keratinocytes have been limited to the immortalized and p53-mutated HaCaT cells and are primarily dependent on BMP6 treatment in mouse epidermal keratinocytes. Despite these insightful analyses, the molecular mechanism underlying the role of BMP signalling in the precise balance between growth arrest and terminal differentiation of keratinocytes still remains not clearly defined. The current study first investigated the hitherto uncharacterized status and functions of BMP signalling in normal human keratinocytes by using three independent strains of primary interfollicular epidermal keratinocytes. Then we provided data demonstrating the role of BMP2 compared to BMP6 in the inhibition of growth and induction of subsequent terminal differentiation of these cells. A second relevant finding is based on the clonal analysis of colony types present in untreated and BMP2/6-treated cultures in absence of EGF. BMP treatment results in the clonal transition from proliferative to abortive colonies, suggesting that BMP signalling most likely inhibits stem cell proliferation and triggers cell cycle exit from transit amplifying cells. Third, we showed evidence that, of the three members of the Cip/Kip family of cyclin-dependent kinase inhibitors, only p57 Kip2 and p21 Cip1 have a BMP2/6-induced expression. One mechanism of inhibition of cell proliferation involves p57 Kip2 as an immediate early response, in contradistinction with p21 Cip1 which largely depends on de novo protein synthesis for its effect to proceed. All together, these results clarify the BMP signalling status in normal primary human keratinocytes and support a new mechanism of inhibition of the proliferation of interfollicular epidermal keratinocytes coupled with induction of their terminal differentiation following BMP2 or BMP6 addition.

Activation of p38- and CRM1-dependent nuclear export promotes E2F1 degradation during keratinocyte differentiation

E2F factors modulate a plethora of cell functions, including proliferation, differentiation, DNA repair and apoptosis. We have shown that differentiation in primary epidermal keratinocytes leads to E2F1 downregulation via activation of protein kinase C and p38 mitogen-activated protein kinase. We now demonstrate that E2F1 downregulation in differentiating keratinocytes involves its ubiquitination, as well as proteasomal degradation subsequent to CRM1-dependent nuclear export. E2F1 nuclear export specifically in response to differentiation requires regions adjacent to the cyclin A-binding domain in the N-terminus of this protein. Significantly, inhibition of p38 interferes with nuclear export and degradation of E2F1 during differentiation, but has no effect on E2F1 in undifferentiated cells. Thus, induction of differentiation in epidermal keratinocytes activates a specific program for post-transcriptional downregulation of E2F1, which involves signaling through p38 and activation of nuclear export pathways.

Expression of Human Beta Defensin 4 in Genetically Modified Keratinocytes Enhances Antimicrobial Activity

Defensins are cationic peptides of the innate host defense system with antimicrobial activity against many of the microorganisms commonly found in burn units. Beta defensins are variably expressed in the epithelia of skin and other organs. Human beta defensin 4 reportedly has antimicrobial activity against Pseudomonas aeruginosa and is not normally expressed in intact skin. Genetic modification was used to ectopically express human beta defensin 4 in cultured primary epidermal keratinocytes. Keratinocytes expressing human beta defensin 4 showed significantly elevated antimicrobial activity against clinically-isolated P. aeruginosa compared with controls. These results suggest that genetic modification of keratinocytes can increase their resistance to microbial contamination. Bioengineered skin replacements containing human beta defensin 4-modified keratinocytes may be useful for transplantation to contaminated burn wounds.

Role of Phagocyte Oxidase in UVA-Induced Oxidative Stress and Apoptosis in Keratinocytes

Chronic exposure to ultraviolet radiation including ultraviolet A (315-400 nm) (UVA) may cause photocarcinogenesis and photoaging. The UVA-induced production of reactive oxygen species (ROS) and the resultant oxidative stress exposure play an important role in these biological processes. Here we have investigated the role of phagocyte oxidase (PHOX, gp91phox) in the production of ROS, redox status change, and apoptosis after UVA exposure by using gp91phox-deficient (gp91phox-/-) primary keratinocytes. UVA radiation resulted in increased ROS production and oxidation of reduced glutathione (GSH) to its oxidized form (GSSG). The presence of diphenylene iodonium (DPI) inhibited ROS production by UVA. In comparison with wild-type cells, gp91phox-/- cells produced slightly less ROS and GSH oxidation. UVA radiation induced apoptosis in wild-type keratinocytes as detected by phosphatidylserine (PS) translocation, caspase activation, and DNA fragmentation. As compared with wild-type cells, UVA induced less PS translocation in gp91phox-deficient cells. No difference, however, was observed in caspase activation and DNA fragmentation after UVA exposure in wild-type and gp91phox-/- cells. These findings suggest that gp91phox plays a limited role in the UVA-induced ROS production, oxidative stress, and therefore the PS translocation, but has no effect on UVA-induced caspase activation and DNA fragmentation during apoptosis.

The effect of arsenicals on ultraviolet-radiation-induced growth arrest and related signaling events in human keratinocytes.

The molecular mechanisms mediating arsenic-induced carcinogenesis are not well understood. The role of confounding factors such as ultraviolet radiation (UV), add another level of complexity to the study of arsenic carcinogenesis and the cancer-risk assessment on humans. We hypothesized that arsenicals are capable of overriding the growth arrest caused by UV treatment and may lead to selective proliferation. To test this hypothesis, a primary normal human epidermal keratinocyte (NHEK) cell culture model was used. One group was pre-exposed to UVB (100 mJ/cm(2)) that arrested a majority ( approximately 95%) of cells in G0/G1 (+UV) and a second group was not exposed to UV (-UV). Treatment of cells with various arsenicals [0-12 microM of inorganic arsenite (iAs), 0-2 microM of methyl oxoarsine (MMAs III) and 0-3 microM of iododimethyl arsine (DMAs III)] indicated a concentration-dependent increase in proliferation at 24 h in the order of DMAs III > MMAs III > iAs. Flow-cytometric analyses revealed differential effects on cell cycle distribution. Analysis of a battery of cell cycle proteins (cyclin D1, cdk5, PCNA, cdc25A and cdc25C) indicated exposure-specific differential expression profiles. Increased activation of JNK phosphorylation (5-10-fold) in the +UV group and the synergistic increase with methyl arsenicals suggested that JNK might be involved in cell survival and proliferative signaling. Induction of EGF levels and increased phosphorylation of the EGF receptor by arsenicals (+UV) suggested that the EGF signaling pathway might mediate arsenical-induced cell proliferation of NHEK cells. Differential activation of ERK1/2 by arsenicals (+/-UV) suggested that EGF-mediated cell proliferation by arsenicals in UV-treated NHEK cells may not involve ERK activation. Taken together, the data suggest that both UV exposure and methylation status of the arsenicals dictate the participation of key cell cycle proteins and related signaling events in arsenical-induced cell proliferation.

Inhibition of Autoantigen Expression by (-)-Epigallocatechin-3-gallate (the Major Constituent of Green Tea) in Normal Human Cells

Autoimmune disorders, characterized by inflammation and apoptosis of target cells leading to tissue destruction, are mediated in part by autoantibodies against normal cellular components (autoantigens) that may be overexpressed. For example, antibodies against the autoantigens SS-A/Ro and SS-B/La are primary markers for systemic lupus erythematosus and Sjögren's syndrome. Recently, studies in animals demonstrated that green tea consumption may reduce the severity of some autoimmune disorders, but the mechanism is unclear. Herein, we sought to determine whether the most abundant green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), affects autoantigen expression in human cells. Cultures of pooled normal human primary epidermal keratinocytes and of an immortalized human salivary acinar cell line were incubated with 100 microM EGCG (a physiologically achievable level for topical application or oral administration) for various time periods and then analyzed by cDNA microarray analysis, reverse transcription-polymerase chain reaction, and Western blotting for expression of several major autoantigen candidates. EGCG inhibited the transcription and translation of major autoantigens, including SS-B/La, SS-A/Ro, coilin, DNA topoisomerase I, and alpha-fodrin. These findings, taken together with green tea's anti-inflammatory and antiapoptotic effects, suggest that green tea polyphenols could serve as an important component in novel approaches to combat autoimmune disorders in humans.

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 (<or=1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and beta-catenin. This is followed by slower (>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.

Suprabasal Induction of Ornithine Decarboxylase in Adult Mouse Skin Is Sufficient to Activate Keratinocytes

To study the effects of de novo induction of ornithine decarboxylase (ODC) activity in adult, quiescent skin, we generated transgenic mice in which the suprabasal expression of an inducible form of the ODC protein fused to a modified estrogen receptor ligand-binding domain (ODCER) is driven by an involucrin promoter. After topical treatment with the inducing agent 4-hydroxytamoxifen (4OHT), ODC activity and putrescine levels were dramatically increased in the epidermis but not in the dermis of transgenic mice. 4OHT treatment stimulated both proliferation as measured by bromodeoxyuridine incorporation in basal epidermal cells and differentiation shown by increased expression of differentiation markers. Furthermore, induction of ODC activity did not rescue primary epidermal keratinocyte cultures isolated from ODCER2 mice from a calcium-triggered DNA synthesis block, as measured by [3H]thymidine incorporation. In vivo induction of epidermal ODC enzyme activity significantly stimulated the vascularization of ODCER transgenic skin. Increased expression of interleukin-1beta and keratin 6, markers of keratinocyte activation seen in wound healing, was also observed in 4OHT-treated transgenic skin. These results suggest that de novo suprabasal induction of ODC activity in adult mouse skin activates keratinocytes and stimulates vascularization in the dermal layer in a manner similar to skin undergoing wound healing.

Bone morphogenetic protein (BMP) signaling controls hair pigmentation by means of cross-talk with the melanocortin receptor-1 pathway.

Hair pigmentation is controlled by tightly coordinated programs of melanin synthesis and involves signaling through the melanocortin type 1 receptor (MC-1R) that regulates the switch between pheomelanogenesis and eumelanogenesis. However, the involvement of other signaling systems, including the bone morphogenetic protein (BMP) pathway, in the control of hair pigmentation remains to be elucidated. To assess the effects of BMP signaling on hair pigmentation, transgenic mice overexpressing the BMP antagonist noggin (promoter: keratin 5) were generated. Whereas wild-type C3H/HeJ mice have a subapical yellow band on otherwise black dorsal hairs, K5-Noggin mice are characterized by the absence of a yellow band and near-black pigment in dorsal coat. Noggin overexpression is accompanied by strongly reduced levels of Agouti signal protein and enhanced expression of microphthalmia transcription factor in the midphase of the hair-growth cycle. Wild-type color in K5-Noggin mice is restored by administration of a synthetic MC-1R antagonist resulting in the reappearance of a subapical yellow band. BMP-4 stimulates the expression of Agouti transcripts and protein in primary epidermal keratinocytes, and BMP signaling positively regulates dermal papilla-specific enhancer of the Agouti gene in primary dermal fibroblasts. Taken together, these data suggests that BMP signaling controls the expression of Agouti protein in the hair follicle and provide evidence for interaction between BMP and MC-1R signaling pathways to modulate the balance between pheomelanogenesis and eumelanogenesis during hair growth.

Phospholipase d signaling and extracellular signal-regulated kinase-1 and -2 phosphorylation (activation) are required for maximal phorbol ester-induced transglutaminase activity, a marker of keratinocyte differentiation.

Protein kinase C (PKC)-activating 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates phospholipase D (PLD) activity in primary mouse epidermal keratinocytes. PLD catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA), which can be dephosphorylated to produce PKC-activating diacylglycerol. In the presence of small amounts of a primary alcohol, PLD can instead produce novel phosphatidylalcohols at the expense of PA and diacylglycerol. Here, we have demonstrated that inhibiting PLD signal generation with 1-butanol reduced TPA-stimulated transglutaminase activity, a marker of keratinocyte differentiation. On the other hand, the structurally related tertiary alcohol tert-butanol, which cannot be used by PLD, had no effect on TPA-induced transglutaminase activity. Since TPA activates all conventional and novel PKC isoforms directly, yet cannot overcome 1-butanol-mediated inhibition, this result suggests that PLD mediates its effects on transglutaminase activity (and keratinocyte differentiation) through an effector enzyme system distinct from the conventional or novel PKC isoenzymes. Data in the literature suggest that PA can recruit Raf-1 to the membrane, where it can be activated and initiate the mitogen-activated protein kinase cascade that culminates in activation of extracellular signal-regulated kinase (ERK)-1 and -2. Indeed, we found that inhibition of ERK-1/2 phosphorylation (activation) inhibited TPA-induced transglutaminase activity. However, inhibition of PLD-mediated signal generation had only a small effect on TPA-elicited ERK-1/2 phosphorylation (activation), whereas inhibition of ERK-1/2 did not affect PLD activation, suggesting that these two pathways likely operate largely in parallel. Thus, our results suggest the independent involvement of the PLD and ERK-1/2 pathways in mediating transglutaminase activity and keratinocyte differentiation.

8-Cl-Adenosine enhances 1,25-dihydroxyvitamin D3-induced growth inhibition without affecting 1,25-dihydroxyvitamin D3-stimulated differentiation of primary mouse epidermal keratinocytes

BackgroundEpidermal keratinocytes continuously proliferate and differentiate to form the mechanical and water permeability barrier that makes terrestrial life possible. In certain skin diseases, these processes become dysregulated, resulting in abnormal barrier formation. In particular, skin diseases such as psoriasis, actinic keratosis and basal and squamous cell carcinomas are characterized by hyperproliferation and aberrant or absent differentiation of epidermal keratinocytes. We previously demonstrated that 8-Cl-adenosine (8-Cl-Ado) can induce keratinocyte growth arrest without inducing differentiation. ResultsTo determine if this agent might be useful in treating hyperproliferative skin disorders, we investigated whether 8-Cl-Ado could enhance the ability of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a known keratinocyte differentiating agent and a clinical treatment for psoriasis, to inhibit keratinocyte growth. We found that low concentrations of 8-Cl-Ado and 1,25(OH)2D3 appeared to act additively to reduce proliferation of primary mouse epidermal keratinocytes. However, another agent (transforming growth factor-beta) that triggers growth arrest without inducing differentiation also coincidentally inhibits differentiation elicited by other agents; inhibition of differentiation is suboptimal for treating skin disorders, as differentiation is often already reduced. Thus, we determined whether 8-Cl-Ado also decreased keratinocyte differentiation induced by 1,25(OH)2D3, as measured using the early and late differentiation markers, keratin 1 protein levels and transglutaminase activity, respectively. 8-Cl-Ado did not affect 1,25(OH)2D3-stimulated keratin 1 protein expression or transglutaminase activity. ConclusionsOur results suggest that 8-Cl-Ado might be useful in combination with differentiating agents for the treatment of hyperproliferative disorders of the skin.

The effects of 1alpha,25-dihydroxyvitamin D3 on the expression of DNA replication genes.

To identify key genes in the antiproliferative action of 1,25(OH)2D3, MC3T3-E1 mouse osteoblasts were subjected to cDNA microarray analyses. Eleven E2F-driven DNA replication genes were downregulated by 1,25(OH)2D3. These results were confirmed by quantitative RT-PCR in different cell types, showing the general nature of this action of 1,25(OH)2D3. 1Alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] has a potent antiproliferative action characterized by a blocked transition from the G1- to the S-phase of the cell cycle. This study aims to identify genes whose expression is markedly altered after 1,25(OH)2D3 treatment in parallel with or preceding the observed G1-arrest. The cDNA microarray technique was used, and the expression of approximately 4600 genes in MC3T3-E1 mouse osteoblasts was studied 6 and 12 h after treatment with 10(-8) M 1,25(OH)2D3. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were performed on MC3T3-E1 cells and on wildtype and vitamin D receptor (VDR) knockout primary murine epidermal keratinocytes (VDRwt MEKs, VDR-/- MEKs) and murine mammary tumor cells (GR) to confirm the microarray data. After 12 h of treatment, in parallel with the 1,25(OH)2D3-induced G1 arrest, a particular set of DNA replication genes including a cell division cycle 6 homolog, a DNA polymerase alpha subunit, proliferating cell nuclear antigen, two DNA polymerase delta subunits, and flap-structure specific endonuclease 1, was downregulated at least 2-fold. These genes are known targets of the E2F family of transcription factors, which are probably the central mediators of this action of 1,25(OH)2D3. Indeed, as shown by transfection assays with an E2F reporter construct, 12- and 24-h treatment of MC3T3-E1 cells with 1,25(OH)2D3 reduced E2F activity by 49% and 73%, respectively. Quantitative RT-PCR analyses confirmed the downregulation of these DNA replication genes by 1,25(OH)2D3 in MC3T3-E1, GR, and VDRwt MEKs cells, but not in VDR-/- MEKs cells, showing that this 1,25(OH)2D3-driven antiproliferative action is of a general nature and depends on a functional VDR.

Tumor Necrosis Factor Related Apoptosis Inducing Ligand Triggers Apoptosis in Dividing but not in Differentiating Human Epidermal Keratinocytes

Using serial analysis of gene expression we have previously identified the expression of several pro-apoptotic and anti-apoptotic genes in cultured human primary epidermal keratinocytes, including tumor necrosis factor related apoptosis inducing ligand (TRAIL). TRAIL is a potent inducer of apoptosis in transformed and tumor cell lines, but usually not in other cells. Here we present a study on the effect of TRAIL on cultured keratinocytes. It is shown that differentiated and undifferentiated keratinocytes undergo apoptosis after addition of TRAIL to the medium as determined by morphologic and biochemical criteria, such as cellular shrinkage and activation of caspases. The sensitivity for TRAIL differs greatly between undifferentiated and differentiating keratinocytes, however, with undifferentiated cells being much more susceptible to apoptosis. Commitment to terminal differentiation in the absence of TRAIL does not in itself induce apoptosis. In contrast to the promyelocytic cell line HL60, internucleosomal DNA fragmentation is not observed in keratinocytes, as assessed by flow cytometric analysis and agarose gel electrophoresis. Interestingly, the prime effector of DNA fragmentation, DNA fragmentation factor of 40 kDa (DFF40), is expressed in keratinocytes, yet internucleosomal cleavage fails to occur. Our data indicate that programmed cell death during keratinocyte differentiation is distinct from receptor-mediated apoptosis in response to a death ligand.