UVB Irradiation Regulates ERK1/2- and p53-Dependent Thrombomodulin Expression in Human Keratinocytes

Thrombomodulin (TM) is highly expressed in endothelial cells and acts as a natural anticoagulation factor to maintain circulation homeostasis. TM is an interesting molecule with many physiological functions, including anti-inflammation, anti-thrombosis, and carcinogenesis inhibition. TM can also be detected on the spinous layer of epidermal keratinocytes. However, the role of epidermal TM is still under investigation. In this study, we investigated keratinocyte TM expression and regulation in response to sub-cytotoxic ultraviolet B (UVB) irradiation. Oxidative stress was assessed with DCF and the results revealed that UVB irradiation significantly and dose-dependently augmented reactive oxygen species (ROS) production in HaCaT cells. In addition, low-dose UVB irradiation decreased TM mRNA and protein levels. Blocking ROS production and ERK activation prevented UVB-induced TM down-regulation. The nuclear p53 accumulation and TM promoter binding was observed within 3 h after UVB exposure. Small interfering RNA-mediated p53 knockdown disrupted the UVB-mediated TM protein down-regulation. Our study demonstrates that UVB irradiation results in ROS accumulation and ERK activation, which causes the nuclear p53 accumulation and TM promoter binding to inhibit TM expression. This study provides novel evidence demonstrating that p53 serves as a key regulator of keratinocyte TM expression.     

Insights into the structural basis of the GADD45beta-mediated inactivation of the JNK kinase, MKK7/JNKK2

NF-kappaB/Rel factors control programmed cell death (PCD), and this control is crucial to oncogenesis, cancer chemoresistance, and antagonism of tumor necrosis factor (TNF) alpha-induced killing. With TNFalpha, NF-kappaB-mediated protection involves suppression of the c-Jun-N-terminal kinase (JNK) cascade, and we have identified Gadd45beta, a member of the Gadd45 family, as a pivotal effector of this activity of NF-kappaB. Inhibition of TNFalpha-induced JNK signaling by Gadd45beta depends on direct targeting of the JNK kinase, MKK7/JNKK2. The mechanism by which Gadd45beta blunts MKK7, however, is unknown. Here we show that Gadd45beta is a structured protein with a predicted four-stranded beta-sheet core, five alpha-helices, and two acidic loops. Association of Gadd45beta with MKK7 involves a network of interactions mediated by its putative helices alpha3 and alpha4 and loops 1 and 2. Whereas alpha3 appears to primarily mediate docking to MKK7, loop 1 and alpha4-loop 2 seemingly afford kinase inactivation by engaging the ATP-binding site and causing conformational changes that impede catalytic function. These data provide a basis for Gadd45beta-mediated blockade of MKK7, and ultimately, TNFalpha-induced PCD. They also have important implications for treatment of widespread diseases.     

Skin mild hypoxia enhances killing of UVB-damaged keratinocytes through reactive oxygen species-mediated apoptosis requiring Noxa and Bim

The naturally occurring skin hypoxia has emerged as a crucial host factor of the epidermal microenvironment. We wanted to systematically investigate how reduced oxygen availability of the epidermis modulates the response of keratinocytes and melanocytes to noxious ultraviolet B radiation (UVB). We report that the exposure of normal human keratinocytes (NHKs) or melanocytes (NHEMs) to mild hypoxia drastically impacts cell death responses following UVB irradiation. The hypoxic microenvironment favors survival and reduces apoptosis of UVB-irradiated NHEMs and their malignant counterparts (melanoma cells). In contrast, NHKs, but not the transformed keratinocytes, under hypoxic conditions display increased levels of reactive oxygen species (ROS) and are significantly sensitized to UVB-mediated apoptosis as compared to NHKs treated under normoxic conditions. Prolonged exposure of UVB-treated NHKs to hypoxia triggers a sustained and reactive oxygen species-dependent activation of the stress kinases p38(MAPK) and JNKs, which in turn, engage the activation of Noxa and Bim proapoptotic proteins. Combined silencing of Noxa and Bim significantly inhibits UVB-mediated apoptosis under hypoxic conditions, demonstrating that hypoxia results in an amplification of the intrinsic apoptotic pathway. Physiologically occurring skin hypoxia, by facilitating the specific removal of UVB-damaged keratinocytes, may represent a decisive host factor impeding important steps of the photocarcinogenesis process.     

Protein Tyrosine Phosphatases,TC-PTP,SHP1, and SHP2, Cooperate in Rapid Dephosphorylation of Stat3 in Keratinocytes Following UVB Irradiation

Stat3 is initially dephosphorylated in murine keratinocytes in response to UVB irradiation. Treatment with Na₃VO₄ desensitized keratinocytes to UVB-induced apoptosis with the recovery of phosphorylated Stat3 protein levels, implying that a protein tyrosine phosphatase (PTP) is involved in this mechanism. In the current work, we report that three PTPs including TC45 (the nuclear form of TC-PTP), SHP1, and SHP2 are involved in this rapid dephosphorylation of Stat3 in keratinocytes induced by UVB irradiation. Dephosphorylation of Stat3 was increased rapidly after UVB irradiation of cultured keratinocytes. Knockdown of TC-PTP, SHP1, or SHP2 using RNAi showed that these PTPs are likely responsible for most of the rapid Stat3 dephosphorylation observed following UVB irradiation. The level of phosphorylated Stat3 was significantly higher in keratinocytes transfected with TC-PTP, SHP1, or SHP2 siRNA in the presence or absence of UVB compared with keratinocytes transfected with control siRNA. TC45 was mainly localized in the cytoplasm of keratinocytes and translocated from cytoplasm to nucleus upon UVB irradiation. Stat3 dephosphorylation was associated with nuclear translocation of TC45. Further studies revealed that knockdown of all three phosphatases, using RNAi, prevented the rapid dephosphorylation of Stat3 following UVB irradiation. In mouse epidermis, the level of phosphorylated Stat3 was initially decreased, followed by a significant increase at later time points after UVB exposure. The levels of Stat3 target genes, such as cyclin D1 and c-Myc, followed the changes in activated Stat3 in response to UVB irradiation. Collectively, these results suggest that three phosphatases, TC45, SHP1, and SHP2, are primarily responsible for UVB-mediated Stat3 dephosphorylation and may serve as part of an initial protective mechanism against UV skin carcinogenesis.     

Activation of T-cell Protein-tyrosine Phosphatase Suppresses Keratinocyte Survival and Proliferation following UVB Irradiation

Chronic exposure to UV radiation can contribute to the development of skin cancer by promoting protein-tyrosine kinase (PTK) signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces protein-tyrosine phosphatase (PTP) inactivation. In the present work, we report that T-cell PTP (TC-PTP) activity is stimulated during the initial response to UVB irradiation, which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. Corroborating with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure, and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer.     

Suppression of apoptosis by UVB irradiation: survival signaling via PI3-kinase/Akt pathway

UVB irradiation induces apoptosis in several cell types. However, we report here that UVB irradiation prevents induction of apoptosis in cells detached from the extracellular matrix under serum-free conditions. NIH3T3 cells cultured in bovine serum albumin-coated dishes (detached from the extracellular matrix) underwent apoptosis under serum-free conditions, which was inhibited by UVB (<0.1 J/cm(2)) irradiation, keeping suspension conditions, as determined by chromatin condensation and the appearance of a subG1 DNA fraction. Furthermore, UVB irradiation decreased caspase-3/7, -8/6, and -9 activation and eliminated loss of mitochondrial inner transmembrane potential, suggesting suppression upstream of the caspase cascade. Treatment with PI3-kinase inhibitors, wortmannin, and LY294002 partly eliminated the UV-mediated inhibition of cell death and recovered the inhibited caspase-3/7 activity. Phosphorylation of Akt was observed from 15 min after UVB irradiation. These results suggested that UVB irradiation transduced a survival signal via PI3 kinase activation and phosphorylation of Akt, and induced some apoptosis inhibition factors upstream of the caspase cascade.     

GADD45b and GADD45g are cdc2/cyclinB1 kinase inhibitors with a role in S and G2/M cell cycle checkpoints induced by genotoxic stress

Gadd45a (Gadd45), Gadd45b (MyD118), and Gadd45g (CR6) constitute a family of evolutionarily conserved, small, acidic, nuclear proteins, which have been implicated in terminal differentiation, growth suppression, and apoptosis. How Gadd45 proteins function in negative growth control is not fully understood. Recent evidence has implicated Gadd45a in inhibition of cdc2/cyclinB1 kinase and in G2/M cell cycle arrest. Yet, whether Gadd45b and/or Gadd45g function as inhibitors of cdc2/cyclinB1 kinase and/or play a role in G2/M cell cycle arrest has not been fully established. In this work, we show that Gadd45b and Gadd45g specifically interact with the Cdk1/CyclinB1 complex, but not with other Cdk/Cyclin complexes, in vitro and in vivo. Data also has been obtained that Gadd45b and Gadd45g, as well as GADD45a, interact with both Cdk1 and cyclinB1, resulting in inhibition of the kinase activity of the Cdk1/cyclinB1 complex. Inhibition of Cdk1/cyclinB1 kinase activity by Gadd45b and Gadd45a was found to involve disruption of the complex, whereas Gadd45g did not disrupt the complex. Moreover, using RKO lung carcinoma cell lines, which express antisense Gadd45 RNA, data has been obtained, which indicates that all three Gadd45 proteins are likely to cooperate in activation of S and G2/M checkpoints following exposure of cells to UV irradiation.     

The enhanced monocyte adhesiveness after UVB exposure requires ROS and NF-kappaB signaling in human keratinocyte

The infiltration of both monocyte and activated T cells in the skin is one of critical steps in the development of UVB-induced inflammation. Upregulation of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) on the surface of keratinocytes plays an important role in this process. In this study, we examined the molecular mechanism responsible for UVB-induced expression of ICAM-1 and subsequent monocyte adhesion by keratinocyte. We observed that (1) UVB induced protein and mRNA expression of ICAM-1 in a dose- and time-dependent manner in human keratinocyte cell HaCaT; (2) UVB induced the translocation of NF-kappaB and inhibition of NF-kappaB by NF-kappaB inhibitors suppressed UVB-induced mRNA and protein expression of ICAM-1; (3) UVB increased the intracellular level of reactive oxygen species (ROS) by HaCaT cells; (4) UVB-induced increase of intracellular ROS level was suppressed by pretreatment with diphenyl iodonium (DPI) and N-acetyl cysteine (NAC); and (5) inhibition of UVB-induced ROS production by DPI or NAC suppressed UVB-mediated translocation of NF-kappaB, expression of ICAM-1 and subsequent monocyte adhesion in HaCaT cells. These results suggest that UVB-induced ROS is involved in the translocation of NF-kappaB which is responsible for expression of ICAM-1 and subsequent increased monocyte adhesion in human keratinocyte.     

Transforming growth factor-beta-induced apoptosis is mediated by Smad-dependent expression of GADD45b through p38 activation

Transforming growth factor-beta (TGF-beta)-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. In this report, we identify GADD45b as an effector of TGF-beta-induced apoptosis. GADD45b has been shown to be a positive mediator of apoptosis induced by certain cytokines and oncogenes. We show that Gadd45b is an immediateearly response gene for TGF-beta and that the proximal Gadd45b promoter is activated by TGF-beta through the action of Smad2, Smad3, and Smad4. We show that ectopic expression of GADD45b in AML12 murine hepatocytes is sufficient to activate p38 and to trigger apoptotic cell death, whereas antisense inhibition of Gadd45b expression blocks TGF-beta-dependent p38 activation and apoptosis. Furthermore, we also show that TGF-beta can activate p38 and induce apoptosis in mouse primary hepatocytes from wild-type mice, but not from Gadd45b-/- mice. All of these findings suggest that GADD45b participates in TGF-beta-induced apoptosis by acting upstream of p38 activation.     

Induction of tissue factor expression in human endothelial cells by CD40 ligand is mediated via activator protein 1, nuclear factor kappa B, and Egr-1

Induction of tissue factor expression in endothelial cells via ligation of CD40 probably figures prominently in the pathogenesis of prevalent inflammatory diseases, including atherosclerosis. However, the molecular mechanisms of tissue factor gene expression triggered by CD40 ligand (CD40L) in this cell type remain unknown. We demonstrate here that the tissue factor promoter region -278 bp to +121 bp contains the CD40L-responsive elements, consisting of activator protein 1 (AP-1)+/-, nuclear factor (NF) kappaB-, and Egr-1-binding sites. Mutations of either the AP-1- or NF-kappaB-binding sites markedly reduced the CD40L-dependent promoter activation. The AP-1 and NF-kappaB sites displayed constitutive and CD40L-enhanceable DNA binding activity, respectively. Of note, mutation of the Egr-1-binding sites, previously not associated with CD40 signaling, impaired activation of the tissue factor promoter. Accordingly, CD40L strongly induced Egr-1 protein expression and DNA binding activity to all three bindings sites. In contrast to CD40L, other established inducers of tissue factor in endothelial cells, interleukin-1beta or tumor necrosis factor alpha, did not increase the expression of Egr-1. In conclusion, induction of tissue factor gene expression in human endothelial cells by CD40L involves AP-1 and NF-kappaB as well as Egr-1, a pathway previously not implicated in CD40 signaling and distinct from that employed by certain other proinflammatory cytokines.     

Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage

Ultraviolet B light (UVB) causes cutaneous inflammation and cell death, but the agents responsible are not defined. These studies examined the role of the platelet-activating factor (PAF) signaling system in UVB-mediated effects. Expression of the PAF receptor in the PAF receptor-negative epidermoid cell line KB augmented apoptosis in response to UVB irradiation. Overexpression of the PAF receptor in primary human keratinocytes also enhanced UVB-mediated apoptosis in vitro, and it enhanced apoptosis in an in vivo model of human keratinocytes grafted onto severe combined immune-deficient (SCID) mice. To define the mechanism by which UVB activates the PAF receptor, we used mass spectrometry to demonstrate significant amounts of the C4 PAF analogs 1-alkyl-2-(butanoyl and butenoyl)-sn-glycero-3-phosphocholine, as well as native PAF in an epidermal cell line after UVB irradiation. Supplementing the cells with the precursor phospholipid 1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (HAPC) increased the amount of C4 PAF analogs recovered after UVB exposure. We irradiated HAPC directly and found, even in the absence of a photosensitizer, fragmentation to C4-PAF receptor ligands. We conclude UVB photo-oxidizes cellular phospholipids, creating PAF analogs that stimulate the PAF receptor to induce further PAF synthesis and apoptosis. PAF signaling may participate in the cutaneous inflammation that occurs during photo-aggravated dermatoses.     

UVB-Induced G2 Arrest of Human Melanocytes Involves Cdc2 Sequestration by Gadd45a in Nuclear Speckles

Exposure to solar UVB radiation is involved in the development of cutaneous melanoma. We previously showed that human melanocytes and melanoma cells respond to UVB radiation via a p53-independent pathway involving GADD45A activation. Here, we determined that UVB-induction of Gadd45a is necessary for G2 arrest and that Gadd45a and its partner p21Waf1 co-localize in nuclear bodies called Nuclear Speckles. We further observed that UVB-induced G2 arrest is associated with Cdc2 accumulation in these Nuclear Speckles. Knock-down of Gadd45a expression by RNA interference prevents both UVB-induced Cdc2 accumulation in Nuclear Speckles and G2 arrest. Our results demonstrate that UVB-induced G2 arrest of melanoma cells is Gadd45a-dependent. Furthermore, we show that Cdc2 sequestration by Gadd45a occurs in Nuclear Speckles, suggesting a new role for these nuclear bodies, so far only linked to RNA maturation.