Shuping Zhong

Involvement of the Acid Sphingomyelinase Pathway in UVA-induced Apoptosis

The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10–40 μm), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20–80 μm), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway.

Mitogen- and Stress-activated Protein Kinase 1 Mediates Activation of Akt by Ultraviolet B Irradiation

In this study, we investigated the mechanism by which UVB irradiation activates Akt (also known as protein kinase B (PKB)) in mouse epidermal JB6 cells. Treatment with a phosphatidylinositol 3-kinase inhibitor, LY 294002, or expression of a dominant negative mutant of p85 (regulatory component of phosphatidylinositol 3-kinase) inhibited UVB-induced Akt activation. Interestingly, Akt activation by UVB was attenuated by treatment with PD 98059, a specific mitogen-activated protein kinase/extracellular signal-regulated protein kinase (Erk) kinase 1 inhibitor, or SB 202190, a specific p38 kinase inhibitor. Furthermore, the expression of a dominant negative mutant of Erk2 or p38 kinase, but not that of c-Jun N-terminal kinase 1 (JNK1), blocked UVB-induced Akt activation. The expression of a dominant negative mutant of p85 or treatment with LY 294002 also inhibited UVB-induced Erk phosphorylation. The UVB-activated mitogen-activated protein kinase members, which were immunoprecipitated from cells exposed to UVB, did not phosphorylate Akt. Instead, Akt was phosphorylated at both threonine 308 and serine 473 and activated by UVB-activated mitogen- and stress-activated protein kinase 1 (Msk1). The expression of a Msk1 C-terminal kinase-dead mutant inhibited UVB-induced phosphorylation and activation of Akt. These data thus suggested that UVB-induced Akt activation was mediated through Msk1, which is a downstream kinase of the Erk and p38 kinase signaling pathways.

PTEN Represses RNA Polymerase III-Dependent Transcription by Targeting the TFIIIB Complex

ABSTRACT PTEN, a tumor suppressor whose function is frequently lost in human cancers, possesses a lipid phosphatase activity that represses phosphatidylinositol 3-kinase (PI3K) signaling, controlling cell growth, proliferation, and survival. The potential for PTEN to regulate the synthesis of RNA polymerase (Pol) III transcription products, including tRNAs and 5S rRNAs, was evaluated. The expression of PTEN in PTEN-deficient cells repressed RNA Pol III transcription, whereas decreased PTEN expression enhanced transcription. Transcription repression by PTEN was uncoupled from PTEN-mediated effects on the cell cycle and was independent of p53. PTEN acts through its lipid phosphatase activity, inhibiting the PI3K/Akt/mTOR/S6K pathway to decrease transcription. PTEN, through the inactivation of mTOR, targets the TFIIIB complex, disrupting the association between TATA-binding protein and Brf1. Kinetic analysis revealed that PTEN initially induces a decrease in the serine phosphorylation of Brf1, leading to a selective reduction in the occupancy of all TFIIIB subunits on tRNALeu genes, whereas prolonged PTEN expression results in the enhanced serine phosphorylation of Bdp1. Together, these results demonstrate a new class of genes regulated by PTEN through its ability to repress the activation of PI3K/Akt/mTOR/S6K signaling.

Phosphorylation at serine 28 and acetylation at lysine 9 of histone H3 induced by trichostatin A.

Trichostatin A (TSA), a histone deacetylase inhibitor, strongly increases acetylation of the N-terminal tails of histone H3. Many studies have correlated the function of TSA with the hyperacetylation of histone. Although histone H3 is known to be phosphorylated, the effect of acetylation on phosphorylation is not known. Here, we report that in JB6 cells, TSA induces both acetylation at lysine 9 and phosphorylation at serine 28 of histone H3. UVB irradiation, which is known to induce phosphorylation at serine 28, did not significantly affect phosphorylation of histone H3 in TSA-pretreated JB6 cells. In contrast, TSA markedly increased phosphorylation and acetylation of histone H3 in UVB-pretreated JB6 cells. TSA strongly activated MAP kinases. Moreover, PD98059 and SB202190 inhibited TSA-induced phosphorylation but not acetylation of histone H3. Dominant negative mutant ERK2 and dominant negative mutant p38 kinase blocked TSA-stimulated phosphorylation of histone H3 at serine 28. The results indicate that TSA-induced phosphorylation of histone H3 at serine 28 occurs through activation of the MAP kinase pathway and phosphorylated histone H3 is more sensitive to TSA-induced hyperacetylation. The facilitation of phosphorylation and acetylation of histone H3 induced by TSA may play a critical regulatory role in chromatin remodeling and gene expression.

Epidermal Growth Factor Enhances Cellular TATA Binding Protein Levels and Induces RNA Polymerase I- and III-Dependent Gene Activity

ABSTRACT TATA binding protein (TBP) is a central transcription factor used by all three cellular RNA polymerases. Changes in the levels of TBP have been shown to have selective effects on gene activity. Overexpression of TBP has been recently shown to contribute to cellular transformation, and elevated levels of TBP occur in a clinically significant proportion of human colon tumors relative to matched normal tissue. To understand the mechanisms by which TBP is regulated, we have analyzed whether activation of the epidermal growth factor receptor (EGFR), a membrane-bound tyrosine receptor kinase that is activated in a large number of human cancers, can serve to regulate cellular TBP. We show that treatment of mouse epidermal cells with EGF produces an increase in TBP levels, which can be blocked with an EGFR-specific inhibitor. In contrast, TBP levels remain unchanged after EGF treatment of EGFR null cells. EGF-mediated increases in TBP are regulated at the transcriptional level, as transient expression of the human TBP promoter is induced with EGF. This regulatory event is dependent upon the downstream activation of Ras and requires the activation of p38, JNK, and ERK mitogen-activated protein kinases. The consequence of elevated TBP on gene expression was further determined. Transcription by RNA polymerase (Pol) I and III was induced by EGF. Directly overexpressing TBP also stimulated transcription from these promoters. Thus, we have identified a new and important target of EGFR signaling, TBP, that contributes to EGF-mediated stimulation of RNA Pol I- and III-dependent gene activity. Since the cellular levels of the products of these genes, tRNAs and rRNAs, determine the translational capacity of cells, this event may be an important contributor to the transforming function of EGF.

The JNKs differentially regulate RNA polymerase III transcription by coordinately modulating the expression of all TFIIIB subunits.

RNA polymerase (pol) III-dependent transcription is subject to stringent regulation by tumor suppressors and oncogenic proteins and enhanced RNA pol III transcription is essential for cellular transformation and tumorigenesis. Since the c-Jun N-terminal kinases (JNKs) display both oncogenic and tumor suppressor properties, the roles of these proteins in regulating RNA pol III transcription were examined. In both mouse and human cells, loss or reduction in JNK1 expression represses RNA pol III transcription. In contrast, loss or reduction in JNK2 expression induces transcription. The JNKs coordinately regulate expression of all 3 TFIIIB subunits. While JNK1 positively regulates TBP expression, the RNA pol III-specific factors, Brf1 and Bdp1, JNK2 negatively regulates their expression. Brf1 is coregulated with TBP through the JNK target, Elk-1. Reducing Elk-1 expression decreases Brf1 expression. Decreasing JNK1 expression reduces Elk-1 occupancy at the Brf1 promoter, while decreasing JNK2 expression enhances recruitment of Elk-1 to the Brf1 promoter. In contrast, regulation of Bdp1 occurs through JNK-mediated alterations in TBP expression. Altered TBP expression mimics the effect of reduced JNK1 or JNK2 levels on Bdp1 expression. Decreasing JNK1 expression reduces the occupancy of TBP at the Bdp1 promoter, while decreasing JNK2 expression enhances recruitment of TBP to the Bdp1 promoter. Together, these results provide a molecular mechanism for regulating RNA pol III transcription through the coordinate control of TFIIIB subunit expression and elucidate opposing functions for the JNKs in regulating a large class of genes that dictate the biosynthetic capacity of cells.

TBP is differentially regulated by c-Jun N-terminal kinase 1 (JNK1) and JNK2 through Elk-1, controlling c-Jun expression and cell proliferation.

ABSTRACT Emerging evidence supports the idea that the c-Jun N-terminal kinases (JNKs) possess overlapping but distinct functions. The potential roles of the ubiquitously expressed JNK1 and JNK2 in regulating expression of the central transcription initiation factor, TATA-binding protein (TBP), were examined. Relative to wild-type fibroblasts, TBP was decreased in Jnk1−/− cells and increased in Jnk2−/− cells. Similarly, reduction of JNK1 in human hepatoma cells decreased TBP expression, whereas reduction of JNK2 enhanced it. JNK-mediated regulation of TBP expression occurs at the transcriptional level through their ability to target Elk-1, which directly regulates the TBP promoter in response to epidermal growth factor stimulation. JNK1 increases, whereas JNK2 decreases, the phosphorylation state of Elk-1, which differentially affects Elk-1 occupancy at a defined site within the TBP promoter. These JNK-mediated alterations in TBP expression, alone, serve to regulate c-Jun expression and fibroblast proliferation rates. These studies uncovered several new molecular events that distinguish the functions of JNK1 and JNK2 that are critical for their regulation of cellular proliferation.

Self-assembled nanoparticles of glycyrrhetic acid-modified pullulan as a novel carrier of curcumin.

Glycyrrhetic acid (GA)-modified pullulan nanoparticles (GAP NPs) were synthesized as a novel carrier of curcumin (CUR) with a degree of substitution (DS) of GA moieties within the range of 1.2–6.2 groups per hundred glucose units. In the present study, we investigated the physicochemical characteristics, release behavior, in vitro cytotoxicity and cellular uptake of the particles. Self-assembled GAP NPs with spherical shapes could readily improve the water solubility and stability of CUR. The CUR release was sustained and pH-dependent. The cellular uptake of CUR-GAP NPs was confirmed by green fluorescence in the cells. An MTT study showed CUR-GAP NPs with higher cytotoxicity in HepG2 cells than free CUR, but GAP NPs had no significant cytotoxicity. GAP is thus an excellent carrier for the solubilization, stabilization, and controlled delivery of CUR.

ERα mediates alcohol-induced deregulation of Pol III genes in breast cancer cells

The association of alcohol consumption and breast cancer is more pronounced in cases that are positive for estrogen receptor (ER+) than in cases that are negative (ER-). Its mechanism remains to be determined. Deregulation of RNA polymerase III (Pol III) transcription enhances cellular tRNAs and 5S rRNA production, increasing translational capacity to promote cell transformation and tumor formation. Here, we report that alcohol increases Pol III gene transcription in both normal and cancer breast cell lines. The induction in ER+ breast cancer cells (MCF-7) is significantly higher than in ER- normal breast cells (MCF-10A, MCF-10F and MCF-12A) and is correlated with ER expression. E2 causes <2-fold increase in Pol III gene transcription. The addition of ethanol to this system now produces a 10-15-fold increase. Ethanol increases ERα expression, resulting in an increase in Brf1 protein and mRNA levels. In addition, ethanol markedly stimulates phosphorylation of JNK1, but not JNK2. Inhibition of JNK1 decreases ERE-Luc reporter activity and represses expression of ERα, Brf1 and Pol III genes. Reduction of ERα by its small interfering RNA represses Brf1 and Pol III gene transcription. Ethanol with E2 produces larger and more numerous colonies. Repression of ERα or Brf1 inhibits alcohol-induced cell transformation. Together, these results support the idea that alcohol increases ERα expression through JNK1 to elevate Brf1 expression and Pol III gene transcription to bring about greater phenotypic changes. These studies demonstrate that ERα mediates Pol III gene transcription through Brf1, suggesting that ERα may play a critical role in alcohol-induced deregulation of Pol III genes in ER+ breast cancer development.

Alcohol Induces RNA Polymerase III-dependent Transcription through c-Jun by Co-regulating TATA-binding Protein (TBP) and Brf1 Expression

Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.