Effects of Individual Amino Acids on PPARα Transactivation, mTORC1 Activation, ApoA-I Transcription and pro-ApoA-I Secretion.

A higher concentration of apolipoprotein A-I (ApoA-I) is associated with increased high density lipoprotein functionality and reverse cholesterol transport (RCT). A promising strategy to prevent cardiovascular diseases is therefore to improve RCT by increasing de novo ApoA-I production. Since experimental animal models have suggested effects of amino acids on hepatic lipoprotein metabolism, we here examined the effects of different amino acids on hepatic ApoA-I production. Human hepatocytes (HepG2) were exposed to six individual amino acids for 48 h. ApoA-I transcription and secreted pro-ApoA-I protein concentrations were analyzed using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assays (ELISA), respectively. Additionally, CPT1 and KEAP1 mRNA expression, peroxisome proliferator-activated receptor alpha (PPARα) transactivation, and mechanistic target of rapamycin complex 1 (mTORC1) phosphorylation were determined. Leucine, glutamic acid, and tryptophan increased ApoA-I and CPT1 mRNA expression. Tryptophan also strongly increased PPARα transactivation. Glutamine, proline, and histidine increased pro-ApoA-I protein concentrations but mTORC1 phosphorylation remained unchanged regardless of the amino acid provided. In conclusion, individual amino acids have different effects on ApoA-I mRNA expression and pro-ApoA-I production which can partially be explained by specific effects on PPARα transactivation, while mTORC1 phosphorylation remained unaffected.

Potential Contribution of Short Chain Fatty Acids to Hepatic Apolipoprotein A-I Production.

Apolipoprotein A-I (ApoA-I) is the major protein of high density lipoprotein (HDL) particles and has a crucial role in reverse cholesterol transport (RCT). It has been postulated that elevating production of de novo ApoA-I might translate into the formation of new functional HDL particles that could lower cardiovascular disease (CVD) risk via RCT. During inflammation, serum ApoA-I concentrations are reduced, which contributes to the development of dysfunctional HDL particles as Serum Amyloid A (SAA) overtakes the position of ApoA-I within the HDL particles. Therefore, instead of elevating serum HDL cholesterol concentrations, rescuing lower serum ApoA-I concentrations could be beneficial in both normal and inflamed conditions. Several nutritional compounds, amongst others short chain fatty acids (SCFAs), have shown their capacity to modulate hepatic lipoprotein metabolism. In this review we provide an overview of HDL and more specific ApoA-I metabolism, SCFAs physiology and the current knowledge regarding the influence of SCFAs on ApoA-I expression and synthesis in human liver cells. We conclude that the current evidence regarding the effect of SCFAs on ApoA-I transcription and secretion is promising, however there is a need to investigate which dietary fibres could lead to increased SCFAs formation and consequent elevated ApoA-I concentrations.

Butyric Acid Added Apically to Intestinal Caco-2 Cells Elevates Hepatic ApoA-I Transcription and Rescues Lower ApoA-I Expression in Inflamed HepG2 Cells Co-Cultured in the Basolateral Compartment.

Apolipoprotein A-I (ApoA-I) concentrations are decreased during inflammation, which may reduce high-density lipoprotein (HDL) functionality. Thus, rescuing ApoA-I concentrations during inflammation might help to prevent atherosclerosis. Recent studies have shown that butyric acid (C4) has anti-inflammatory effects and rescues ApoA-I production. However, whether intestinal short chain fatty acids (SCFAs) are able to influence hepatic processes is unknown. Therefore, we investigated C4 anti-inflammatory effects on ApoA-I transcription in the intestine-liver co-culture model. C4 dose-response experiments in the presence or absence of cytokines were performed in a co-culture system including Caco-2 cells, HepG2 cells, or both. Changes in ApoA-I transcription in Caco-2 cells and HepG2 cells were analyzed using qPCR. C4 increased ApoA-I expression in HepG2 cells that cultured alone. When both cells were cultured together, C4 decreased ApoA-I expression in Caco-2 cells and increased ApoA-I expression in HepG2 cells. However, adding C4 to apical Caco-2 cells resulted in a smaller effect in HepG2 cells compared with adding C4 directly to the hepatocytes. Moreover, C4 rescued ApoA-I expression in inflamed HepG2 cells. These findings suggests that intestinal SCFAs can affect hepatic processes. However, the smaller effect in the co-culture experiment indicates cross-talk between intestine and liver.

Short-Chain Fatty Acids (Except Hexanoic Acid) Lower NF-kB Transactivation, Which Rescues Inflammation-Induced Decreased Apolipoprotein A-I Transcription in HepG2 Cells.

Concentrations of apolipoprotein A-I (ApoA-I) decrease during inflammation, which may lead to dysfunctional ApoA-I-poor high-density lipoprotein (HDL) particles, and as such, elevate cardiovascular risk. Therefore, rescuing ApoA-I concentrations, especially during inflammation, seems beneficial. Recently, short-chain fatty acids (SCFAs) have received more attention as a strategy in reversing atherosclerosis. We here evaluated the effects of SCFAs on inflammatory pathways in relation to ApoA-I transcription. SCFAs dose-response studies were performed in the presence and absence of inflammatory cytokines. ApoA-I and interleukin 8 (IL-8) mRNA expression were analyzed using qPCR and ELISA, respectively. To study underlying mechanisms, nuclear factor kappa B (NF-κB) transactivation and changes in mRNA expressions of the genes targets of bromodomain and extra-terminal (BET) inhibition, peroxisome proliferator-activated receptor-alpha (PPARα) transactivation and activator protein 1 (AP-1) pathway were analyzed. SCFAs (except hexanoic acid) increased ApoA-I mRNA transcription in both normal and inflammatory conditions and lowered IL-8 mRNA expression. This anti-inflammatory effect of SCFAs was confirmed by inhibition of NF-κB transactivation. Moreover, butyric acid increased carnitine palmitoyltransferase 1 (CPT1), PPARα target gene, mRNA transcription in both conditions, and there was a negative correlation between CPT1 and NF-κB. Therefore, PPARα transactivation is probably involved in the anti-inflammatory effects of SCFAs, which rescues ApoA-I transcription. In conclusion, propionate, butyrate and valerate elicit anti-inflammatory effects which might rescue ApoA-I transcription in inflammatory conditions via PPARα transactivation mediated NF-κB inhibition.

Search for Natural Compounds That Increase Apolipoprotein A-I Transcription in HepG2 Cells: Specific Attention for BRD4 Inhibitors.

Although increasing apolipoprotein A-I (apoA-I) might lower the cardiovascular disease risk, knowledge on natural compounds that elevate apoA-I transcription is limited. Therefore, the aim of this study was to discover natural compounds that increase apoA-I transcription in HepG2 cells. Since BRD4 inhibition is known to elevate apoA-I transcription, we focused on natural BRD4 inhibitors. For this, the literature was screened for compounds that might increase apoA-I and or inhibit BRD4. This resulted in list A, (apoA-I increasers with unknown BRD4 inhibitor capacity), list B (known BRD4 inhibitors that increase apoA-I), and list C (BRD4 inhibitors with unknown effect on apoA-I). These compounds were compared with the compounds in two natural compound databases. This resulted in (1) a common substructure (ethyl-benzene) in 60% of selected BRD4-inhibitors, and (2) four compounds that increased ApoA-I: hesperetin, equilenin, 9(S)-HOTrE, and cymarin. Whether these increases are regulated via BRD4 inhibition and the ethyl-benzene structure inhibits BRD4 requires further study.

Amoxicillin Modulates ApoA-I Transcription and Secretion, Predominantly via PPARα Transactivation Inhibition.

In a recent human study, we observed that amoxicillin treatment decreased HDL-C concentration. We hypothesize that antibiotics lower the transcription and secretion of ApoA-I, the responsible protein for HDL production. HepG2 and Caco-2 cells were exposed to increasing dose of amoxicillin, penicillin, and streptomycin. Secreted ApoA-I protein and mRNA transcripts were analyzed using ELISA and qPCR, respectively. To unravel underlying mechanisms, KEAP1, CPT1, and CHOP mRNA expressions were determined as well as PPARα transactivation. In HepG2 and Caco-2, amoxicillin decreased ApoA-I transcription and secretion. Effects on ApoA-I expression were clearly there for amoxicillin while no effects were observed for penicillin or streptomycin. KEAP1, CPT1, and CHOP mRNA expressions were reduced by amoxicillin treatments. Moreover, a significant correlation between ApoA-I and CPT1 mRNA expressions was found. Furthermore, amoxicillin lowered PPARα transactivation. All together, these data suggest that inhibited PPARα transactivation is involved in the effects of amoxicillin on ApoA-I. In conclusion, the direct effect of amoxicillin in treated HepG2 and Caco-2 cells was a lower ApoA-I secretion and transcription. Based on evaluating alterations in KEAP1, CPT1, and CHOP mRNA expressions plus PPARα transactivation, we suggest that a reduced PPARα activation is a potential mechanism behind the observed amoxicillin effects on ApoA-I expression.

JNK-Dependent cJun Phosphorylation Mitigates TGFß- and EGF-Induced Pre-Malignant Breast Cancer Cell Invasion by Suppressing AP-1-Mediated Transcriptional Responses.

Transforming growth factor-ß (TGFß) has both tumor-suppressive and tumor-promoting effects in breast cancer. These functions are partly mediated through Smads, intracellular transcriptional effectors of TGFß. Smads form complexes with other DNA-binding transcription factors to elicit cell-type-dependent responses. Previously, we found that the collagen invasion and migration of pre-malignant breast cancer cells in response to TGFß and epidermal growth factor (EGF) critically depend on multiple Jun and Fos components of the activator protein (AP)-1 transcription factor complex. Here we report that the same process is negatively regulated by Jun N-terminal kinase (JNK)-dependent cJun phosphorylation. This was demonstrated by analysis of phospho-deficient, phospho-mimicking, and dimer-specific cJun mutants, and experiments employing a mutant version of the phosphatase MKP1 that specifically inhibits JNK. Hyper-phosphorylation of cJun by JNK strongly inhibited its ability to induce several Jun/Fos-regulated genes and to promote migration and invasion. These results show that MEK-AP-1 and JNK-phospho-cJun exhibit distinct pro- and anti-invasive functions, respectively, through differential regulation of Smad- and AP-1-dependent TGFß target genes. Our findings are of importance for personalized cancer therapy, such as for patients suffering from specific types of breast tumors with activated EGF receptor-Ras or inactivated JNK pathways.

The effects of short-chain fatty acids on the transcription and secretion of apolipoprotein A-I in human hepatocytes in vitro.

BACKGROUND: Apolipoprotein-I (ApoA-I), the major component of high-density lipoprotein (HDL) particles, mediates cholesterol efflux by which it facilitates the removal of excess cholesterol from peripheral tissues. Therefore, elevating ApoA-I production leading to the production of new pre-ß-HDL particles is thought to be beneficial in the prevention of cardiovascular diseases. Recently, we observed that amoxicillin treatment led to decreased HDL concentrations in healthy human volunteers. We questioned whether this antibiotic effect was directly or indirectly, via changed short-chain fatty acids (SCFA) concentrations through an altered gut microflora. Therefore, we here evaluated the effects of amoxicillin and various SCFA on hepatic ApoA-I expression, secretion, and the putative underlying pathways. METHODS AND RESULTS: Human hepatocytes (HepG2) were exposed to increasing dose of amoxicillin or SCFA for 48 hours. ApoA-I messenger RNA (mRNA) transcription and secreted protein were analyzed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. To study underlying mechanisms, changes in mRNA expression of KEAP1, CPT1, and PPARα, as well as a PPARα transactivation assay, were analyzed. Amoxicillin dose-dependently decreased ApoA-I mRNA transcription as well as ApoA-I protein secretion. SCFA treatment resulted in a dose-dependent stimulation of ApoA-I mRNA transcription, however, the ApoA-I protein secretion was decreased. Furthermore, SCFA treatment increased PPARα transactivation, PPARα and CPT1 mRNA transcription, whereas KEAP1 mRNA transcription was decreased. CONCLUSION: Direct treatment of HepG2 cells with amoxicillin has either direct effects on lowering ApoA-I transcription and secretion or indirect effects via modified SCFA concentrations because SCFA were found to stimulate hepatic ApoA-I expression. Furthermore, BET inhibition and PPARα activation were identified as possible mechanisms behind the observed effects on ApoA-I transcription.

Large-Scale Screening of Natural Products Transactivating Peroxisome Proliferator-Activated Receptor α Identifies 9S-Hydroxy-10E,12Z,15Z-Octadecatrienoic Acid and Cymarin as Potential Compounds Capable of Increasing Apolipoprotein A-I Transcription in Human Liver Cells.

Increasing apolipoprotein A-I (apoA-I), the predominant protein of high-density lipoprotein (HDL) particles, has favorable effects on atherogenic risk factors. Here, we investigated the effects of peroxisome proliferator-activated receptor α (PPARα) transactivating compounds on apoA-I transcription in HepG2 cells. A transient PPARα agonist transactivation assay was used to screen 2500 natural compounds. To analyze the effects on apoA-I transcription, human hepatocellular liver carcinoma (HepG2) were exposed to 0.1, 1, and 10 µg/mL of the natural PPARα transactivators. ApoA-I mRNA expression was determined by quantitative polymerase chain reaction. Extensive dose-response experiments were performed using compounds that increased apoA-I transcription by minimally 20%. Kelch-like ECH-associated protein 1 (KEAP) and carnitine palmitoyltransferase 1 alpha (CPT1α) expression were used respectively to confirm Bromodomain-containing protein 4 inhibition or PPARα activation. Twenty-eight natural compounds increased PPARα transactivation by at least twofold. Despite the increased CPT1α expression seen after the addition of most PPARα activating compounds, CPT1α expression and PPARα transactivation did not correlate. Addition of 0.05 µg/mL 9S-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9(S)-HOTrE) increased apoA-I mRNA expression by 35%, whereas 10-25 µg/mL of cymarin increased apoA-I transcription by 37%. However, combining cymarin and 9(S)-HOTrE did not result in a synergistic effect, in contrast this combination even decreased apoA-I transcription. ApoA-I transcription involves multiple regulatory players, and PPARα transactivation alone is not sufficient. A search for natural compounds resembling the molecular structure of 9(S)-HOTrE or cymarin could aid to find additional components that increase apoA-I transcription.

C/EBP-ß Is Differentially Affected by PPARα Agonists Fenofibric Acid and GW7647, But Does Not Change Apolipoprotein A-I Production During ER-Stress and Inflammation.

Increasing apolipoproteinA-I (apoA-I) production may be anti-atherogenic. Thus, there is a need to identify regulatory factors involved. Transcription of apoA-I involves peroxisome-proliferator-activated-receptor-alpha (PPARα) activation, but endoplasmic reticulum (ER) -stress and inflammation also influence apoA-I production. To unravel why PPARα agonist GW7647 increased apoA-I production compared to PPARα agonist fenofibric acid (FeAc) in human hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (CaCo-2) cells, gene expression profiles were compared. Microarray analyses suggested CCAAT/enhancer-binding-protein-beta (C/EBP-ß) involvement in the FeAc condition. Therefore, C/EBP-ß silencing and isoform-specific overexpression experiments were performed under ER-stressed, inflammatory and non-inflammatory conditions. mRNA expression of C/EBP-ß, ATF3, NF-IL3 and GDF15 were upregulated by FeAc compared to GW7647 in both cell lines, while DDIT3 and DDIT4 mRNA were only upregulated in HepG2 cells. This ER-stress related signature was associated with decreased apoA-I secretion. After ER-stress induction by thapsigargin or FeAc addition, intracellular apoA-I concentrations decreased, while ER-stress marker expression (CHOP, XBP1s, C/EBP-ß) increased. Cytokine addition increased intracellular C/EBP-ß levels and lowered apoA-I concentrations. Although a C/EBP binding place is present in the apoA-I promoter, C/EBP-ß silencing or isoform-specific overexpression did not affect apoA-I production in inflammatory, non-inflammatory and ER-stressed conditions. Therefore, C/EBP-ß is not a target to influence hepatic apoA-I production. J. Cell. Biochem. 118: 754-763, 2017. © 2016 Wiley Periodicals, Inc.

Link Between ER-Stress, PPAR-Alpha Activation, and BET Inhibition in Relation to Apolipoprotein A-I Transcription in HepG2 Cells.

Activating transcription factor peroxisome proliferator-activated receptor alpha (PPARα) may increase apoA-I transcription. Furthermore, Bromodomain and Extra-Terminal domain (BET) protein inhibitors increase, whereas Endoplasmic Reticulum (ER) stress decreases apoA-I transcription. We examined possible links between these processes as related to apoA-I transcription in HepG2 cells. JQ1(+), thapsigargin, and GW7647 were used to induce, respectively BET inhibition, ER-stress, and PPARα activation. Expression of ER-stress markers (CHOP, XBP1s) was analyzed by western blotting. PPARα, KEAP1 (marker for BET inhibition), and apoA-I mRNAs were measured using qPCR. ER-stress and BET inhibition both decreased PPARα mRNA expression and activity, but did not interfere with each other, as ER-stress did not change KEAP1 and JQ1(+) did not influence ER-stress marker production. Interestingly, PPARα activation and BET-inhibition diminished ER-stress marker production and rescued apoA-I transcription during existing ER-stress. We conclude that the ER-stress mediated reduction in apoA-I transcription could be partly mediated via the inhibition of PPARα mRNA expression and activity. In addition, BET inhibition increased apoA-I transcription, even if PPARα production and activity were decreased. Finally, both BET inhibition and PPARα activation ameliorate the apoA-I lowering effect of ER-stress and are therefore interesting targets to elevate apoA-I transcription. J. Cell. Biochem. 118:2161-2167, 2017. © 2016 Wiley Periodicals, Inc.

HLA-E regulates NKG2C+ natural killer cell function through presentation of a restricted peptide repertoire.

UNLABELLED: NK cells interact with the HLA-E molecule via the inhibitory receptor NKG2A and the activating receptor NKG2C. Hence, HLA-E can have a dual role in the immune response. In the present study, we aim to investigate the functional consequences of HLA-E for NKG2A and NKG2C expressing NK cell subsets by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. PBMC derived from healthy subjects were used as targets for isolated NK cells and NK cell activation was examined by analysis of the expression of the degranulation marker CD107a. Peptide induced HLA-E expression inhibited degranulation of NKG2A+ NK cell subsets with almost all peptides, whereas NKG2A- NKG2C+ NK cell responses were enhanced only after incubation with four peptides; 1.3-fold with CMV(I), A80 and B13 and 3.2-fold with HLA-G derived peptide. In addition, the HLA-E:G peptide complex triggered NKG2C receptor internalization, as evidenced by reduction in the percentage of NKG2C+ NK cells when incubated with the peptide, which could be restored by addition of Bafilomycin. IN CONCLUSION: in contrast to NKG2A, NKG2C is regulated by HLA-E only when HLA-E is in complex with a restricted peptide repertoire, especially in combination with the HLA-G leader peptide.

CCAAT/enhancer binding protein ß in relation to ER stress, inflammation, and metabolic disturbances.

The prevalence of the metabolic syndrome and underlying metabolic disturbances increase rapidly in developed countries. Various molecular targets are currently under investigation to unravel the molecular mechanisms that cause these disturbances. This is done in attempt to counter or prevent the negative health consequences of the metabolic disturbances. Here, we reviewed the current knowledge on the role of C/EBP-ß in these metabolic disturbances. C/EBP-ß deletion in mice resulted in downregulation of hepatic lipogenic genes and increased expression of ß-oxidation genes in brown adipose tissue. Furthermore, C/EBP-ß is important in the differentiation and maturation of adipocytes and is increased during ER stress and proinflammatory conditions. So far, studies were only conducted in animals and in cell systems. The results found that C/EBP-ß is an important transcription factor within the metabolic disturbances of the metabolic system. Therefore, it is interesting to examine the potential role of C/EBP-ß at molecular and physiological level in humans.

Fatty acid chain length and saturation influences PPARα transcriptional activation and repression in HepG2 cells.

SCOPE: Fatty acids regulate peroxisome proliferator activated receptor α (PPARα) activity, however, most studies evaluated the binding ability of fatty acids to PPARα, which does not necessarily result in PPARα transactivation. We therefore examined dose-response relationships between fatty acids and PPARα transactivation in HepG2 cells. Secretion of apoA-I protein as well as CPT1, ACO, and PPARα mRNA expression, all accepted PPARα targets, were determined as read-outs. METHODS AND RESULTS: HepG2 cells transfected with full-length human PPARα and a PPAR response element luciferase reporter were exposed to different fatty acid concentrations. Lauric and lower doses of myristic acid increased PPARα transactivation. Palmitic and stearic acid inhibited and their monounsaturated counterparts, palmitoleic and oleic acid, increased PPARα transactivation. Linoleic and γ-linolenic acid did not influence PPARα transactivation, while α-linolenic acid strongly increased transactivation. Arachidonic, eicosapentaenoic acid, and docosahexaenoic acid all activated PPARα transactivation at lower doses, but acted at higher concentrations as PPARα repressors. In line with these results, α-linolenic acid increased and docosahexaenoic acid decreased apoA-I protein secretion and PPARα mRNA expression. Interestingly, ACO mRNA expression did not change while CPT1 mRNA expression showed the opposite pattern. CONCLUSION: We found that fatty acids, reported to bind strongly to PPARα, could even repress PPARα transactivation illustrating that these binding assays should be interpreted with caution.

Midterm peer feedback in problem-based learning groups: the effect on individual contributions and achievement.

Even though peer process feedback is an often used tool to enhance the effectiveness of collaborative learning environments like PBL, the conditions under which it is best facilitated still need to be investigated. Therefore, this study investigated the effects of individual versus shared reflection and goal setting on students' individual contributions to the group and their academic achievement. In addition, the influence of prior knowledge on the effectiveness of peer feedback was studied. In this pretest-intervention-posttest study 242 first year students were divided into three conditions: condition 1 (individual reflection and goal setting), condition 2 (individual and shared reflection and goal setting), and condition 3 (control group). Results indicated that the quality of individual contributions to the tutorial group did not improve after receiving the peer feedback, nor did it differ between the three conditions. With regard to academic achievement, only males in conditions 1 and 2 showed better academic achievement compared with condition 3. However, there was no difference between both ways of reflection and goal setting with regard to achievement, indicating that both ways are equally effective. Nevertheless, it is still too early to conclude that peer feedback combined with reflection and goal setting is not effective in enhancing students' individual contributions. Students only had a limited number of opportunities to improve their contributions. Therefore, future research should investigate whether an increase in number of tutorial group meetings can enhance the effectiveness of peer feedback. In addition, the effect of quality of reflection and goal setting could be taken into consideration in future research.

Amino acids stimulate Akt phosphorylation, and reduce IL-8 production and NF-κB activity in HepG2 liver cells.

Hepatic insulin resistance and inflammatory cytokine production contribute to the manifestation of the metabolic syndrome. As amino acids have been implicated in modulating insulin signaling and inflammation, we have investigated the effects of glutamine, leucine and proline on markers of inflammation and insulin sensitivity in HepG2 liver cells. Cells were incubated with IL-1ß (5 ng/mL) to stimulate IL-8 production. After 24 h, glutamine inhibited IL-8 production significantly (p<0.05) at 2, 5 and 10 mM (to 82, 73 and 72% of control), whereas leucine reduced IL-8 production significantly only at 10 mM (66%) and proline at 5 and 10 mM (71 and 52%). Glutamine, leucine and proline all reduced NF-κB activity after 3 h of IL-1ß stimulation at 2, 5 and 10 mM (p<0.001). Insulin-induced (1 nM) Akt phosphorylation was reduced in cells treated with tumour necrosis factor-α (10 ng/mL) for 24 h, but was partly restored by simultaneous incubation with glutamine, leucine and proline (25 mM). Phosphorylation of glycogen synthase kinase-3ß was unaffected by insulin stimulation and amino acid treatment. Our results indicate that glutamine, leucine and proline attenuate IL-8 production, probably through inhibition of NF-κB, and that they increase Akt phosphorylation in HepG2 cells.

ATF3 and Fra1 have opposite functions in JNK- and ERK-dependent DNA damage responses.

JNK and ERK MAP kinases regulate cellular responses to genotoxic stress in a cell type and cell context-dependent manner. However, the factors that determine and execute JNK- and ERK-controlled stress responses are only partly known. In this study, we investigate the roles of the AP-1 components ATF3 and Fra1 in JNK- and ERK-dependent cell cycle arrest and apoptosis. We show that the anti-cancer drug cisplatin or UV light activates both JNK and ERK in human glioblastoma cells lacking functional p53. Inhibition experiments of JNK or ERK activities revealed that the ERK pathway strongly promotes cisplatin- and UV-induced apoptosis in these glioblastoma cells. Furthermore, JNK but not ERK is required for ATF3 induction, and both ERK and JNK are necessary for post-transcriptional induction of Fra1 in response to cisplatin or UV. Knock-down of ATF3 and Fra1 results in increased and decreased cisplatin-induced apoptosis, respectively, indicating that ATF3 is an anti-apoptotic JNK effector and Fra1 is a pro-apoptotic ERK/JNK effector. Knock-down experiments also revealed that ATF3 and Fra1, respectively, enhance and reduce S-phase arrest through differential modulation of the Chk1-Cdk2 pathway. Thus, we identify novel reciprocal functions of ATF3 and Fra1 in JNK- and ERK-dependent DNA damage responses.

DNA damage in transcribed genes induces apoptosis via the JNK pathway and the JNK-phosphatase MKP-1.

The nucleotide excision repair (NER) system consists of two sub-pathways, global genome repair (GGR) and transcription-coupled repair (TCR), which exhibit distinct functions in the cellular response to genotoxic stress. Defects in TCR result in prolonged UV light-induced stalling of RNA polymerase II and hypersensitivity to apoptosis induced by UV and certain chemotherapeutic drugs. Here, we show that low doses of UV trigger delayed activation of the stress-induced MAPkinase JNK and its proapoptotic targets c-Jun and ATF-3 in TCR-deficient primary human fibroblasts from Xeroderma Pigmentosum (XP) and Cockayne syndrome (CS) patients. This delayed activation of the JNK pathway is not observed in GGR-deficient TCR-proficient XP cells, is independent of functional p53, and is established through repression of the JNK-phosphatase MKP-1 rather than by activation of the JNK kinases MKK4 and 7. Enzymatic reversal of UV-induced cyclobutane pyrimidine dimers (CPDs) by CPD photolyase abrogated JNK activation, MKP-1 repression, and apoptosis in TCR-deficient XPA cells. Ectopic expression of MKP-1 inhibited DNA-damage-induced JNK activity and apoptosis. These results identify both MKP-1 and JNK as sensors and downstream effectors of persistent DNA damage in transcribed genes and suggest a link between the JNK pathway and UV-induced stalling of RNApol II.