Kaempferide Prevents Photoaging of Ultraviolet-B Irradiated NIH-3T3 Cells and Mouse Skin via Regulating the Reactive Oxygen Species-Mediated Signalings.

Kaempferide (KFD) is a naturally occurring flavonoid that exists in various medicinal plants. The pharmaceutical properties of KFD, including its anti-cancer, antioxidant and anti-diabetic effects, have been noted, but the effects of KFD on photoaging and their underlying molecular mechanism have yet to be elucidated. In this study, we investigated the effects of KFD on Ultraviolet-B (UVB)-mediated photoaging processes using in vitro and in vivo photoaging model systems. The topical administration of KFD on mouse dorsal areas suppressed UVB-mediated wrinkle formation and epidermal thickening. In addition, the UVB-mediated reduction of dermal collagen content, which was estimated by Masson's trichrome staining, was recovered through KFD treatments. Furthermore, we found that UVB-induced abnormal values of procollagen type-1 (COL1A1), metalloproteinases (MMP-1a and MMP-3) and proinflammatory cytokines (IL-8, MCP-3 and IL-6) on mouse skin tissue as well as NIH-3T3 cells was recovered through KFD treatment. The administration of KFD to NIH-3T3 cells suppressed the UVB-mediated upregulation of reactive oxygen species (ROS), mitogen-activated protein kinases (MAPKs) and AKT phosphorylation. Furthermore, the treatment of ROS inhibitor restored the UVB-induced MAPKs and AKT phosphorylation as well as the abnormal expression of photoaging related genes. These findings indicate that KFD can attenuate UVB-induced ROS elevation to elicit anti-photoaging activity. Taken together, our data suggest that KFD could be developed as a potential natural anti-photoaging agent.

Murine Model of Cardiac Defects Observed in Adams-Oliver Syndrome Driven by Delta-Like Ligand-4 Haploinsufficiency.

Heterozygous loss-of-function mutation in Delta-like ligand-4 (Dll4) is an important cause of Adams-Oliver syndrome (AOS). Cardiac defects, in particular outflow tract (OFT) alignment defects, are observed in about one-fourth of patients with this syndrome. The mechanism underlying this genotype-phenotype correlation has not yet been established. Dll4-mediated Notch signaling is known to play a crucial role in second heart field (SHF) progenitor cell proliferation. We hypothesized that the depletion of the SHF progenitor pool of cells due to partial loss of Dll4 is responsible for the OFT alignment defects seen in AOS. To demonstrate this, we studied Dll4 expression by murine SHF progenitor cells around E9.5, a crucial time-point in SHF biology. We used SHF-specific (Islet1-Cre) conditional knockout of Dll4 to bypass the early embryonic lethality seen in global Dll4 heterozygotes. Dll4-mediated Notch signaling is critically required for SHF proliferation such that Dll4 knockout results in a 33% reduction in proliferation and a fourfold increase in apoptosis in SHF cells, leading to a 56% decline in the size of the SHF progenitor pool. A reduction in SHF cells available for incorporation into the developing heart leads to underdevelopment of the SHF-derived right ventricle and OFT. Similar to the clinical syndrome, 32% of SHF-specific Dll4 heterozygotes demonstrate foreshortened and misaligned OFT, resulting in a double outlet right ventricle. Our murine model provides a molecular mechanism to explain the cardiac defects observed in AOS and establishes a novel clinical role for Dll4-mediated Notch signaling in SHF progenitor biology.

Loss of muscleblind-like 1 results in cardiac pathology and persistence of embryonic splice isoforms.

Cardiac dysfunction is a prominent cause of mortality in myotonic dystrophy I (DM1), a disease where expanded CUG repeats bind and disable the muscleblind-like family of splice regulators. Deletion of muscleblind-like 1 (Mbnl1(ΔE2/ΔE2)) in 129 sv mice results in QRS, QTc widening, bundle block and STc narrowing at 2-4 months of age. With time, cardiac function deteriorates further and at 6 months, decreased R wave amplitudes, sinus node dysfunction, cardiac hypertrophy, interstitial fibrosis, multi-focal myocardial fiber death and calcification manifest. Sudden death, where no end point illness is overt, is observed at a median age of 6.5 and 4.8 months in ~67% and ~86% of male and female Mbnl1(ΔE2/ΔE2) mice, respectively. Mbnl1 depletion results in the persistence of embryonic splice isoforms in a network of cardiac RNAs, some of which have been previously implicated in DM1, regulating sodium and calcium currents, Scn5a, Junctin, Junctate, Atp2a1, Atp11a, Cacna1s, Ryr2, intra and inter cellular transport, Clta, Stx2, Tjp1, cell survival, Capn3, Sirt2, Csda, sarcomere and cytoskeleton organization and function, Trim55, Mapt, Pdlim3, Pdlim5, Sorbs1, Sorbs2, Fhod1, Spag9 and structural components of the sarcomere, Myom1, Tnnt2, Zasp. Thus this study supports a key role for Mbnl1 loss in the initiation of DM1 cardiac disease.

Gene dysregulation by histone variant H2A.Z in bladder cancer.

BACKGROUND: The incorporation of histone variants into nucleosomes is one of the main strategies that the cell uses to regulate the structure and function of chromatin. Histone H2A.Z is an evolutionarily conserved histone H2A variant that is preferentially localized within nucleosomes at the transcriptional start site (TSS). H2A.Z reorganizes the local chromatin structure and recruits the transcriptional machinery for gene activation. High expression of H2A.Z has been reported in several types of cancers and is causally linked to genomic instability and tumorigenesis. However, it is not entirely clear how H2A.Z overexpression in cancer cells establishes aberrant chromatin states and promotes gene expression. RESULTS: Through integration of genome-wide H2A.Z ChIP-seq data with microarray data, we demonstrate that H2A.Z is enriched around the TSS of cell cycle regulatory genes in bladder cancer cells, and this enrichment is correlated with the elevated expression of cancer-promoting genes. RNAi-mediated knockdown of H2A.Z in the cancer cells causes transcriptional suppression of multiple cell cycle regulatory genes with a distinct decrease in cell proliferation. H2A.Z nucleosomes around the TSS have higher levels of H3K4me2/me3, which coincides with the recruitment of two chromatin factors, WDR5 and BPTF. The observed recruitment is functional, as the active states of H2A.Z target genes are largely erased by suppressing the expression of WDR5 or BPTF, effects resembling H2A.Z knockdown. CONCLUSIONS: We conclude that H2A.Z is overexpressed in bladder cancer cells and contributes to cancer-related transcription pathways. We also provide evidence in support of the engagement of H3K4me2/me3 and WDR5/BPTF in H2A.Z-induced cancer pathogenesis. Further studies are warranted to understand how H2A.Z overexpression contributes to the recruitment of the full repertoire of transcription machinery to target genes in bladder cancer cells.

VprBP has intrinsic kinase activity targeting histone H2A and represses gene transcription.

Histone modifications play important roles in the regulation of gene expression and chromatin organization. VprBP has been implicated in transcriptionally silent chromatin formation and cell-cycle regulation, but the molecular basis underlying such effects remains unclear. Here we report that VprBP possesses an intrinsic protein kinase activity and is capable of phosphorylating histone H2A on threonine 120 (H2AT120p) in a nucleosomal context. VprBP is localized to a large set of tumor suppressor genes and blocks their transcription, in a manner that is dependent on its kinase activity toward H2AT120. The functional significance of VprBP-mediated H2AT120p is further underscored by the fact that RNAi knockdown and small-molecule inhibition of VprBP reactivate growth regulatory genes and impede tumor growth. Our findings establish VprBP as a major kinase responsible for H2AT120p in cancer cells and suggest that VprBP inhibition could be a new strategy for the development of anticancer therapeutics.

p53 requires an intact C-terminal domain for DNA binding and transactivation.

The tumor suppressor p53 plays a critical role in mediating cellular response to a wide range of environmental stresses. p53 regulates these processes mainly by acting as a short-lived DNA binding protein that stimulates transcription from numerous genes involved in cell cycle arrest, programmed cell death, and other processes. To investigate the importance of the C-terminal domain of p53, we generated a series of deletion and point mutations in this region and analyzed their effects on p53 transcription activity. Our results show that C-terminal deletion and point mutations at K320 and K382 abolish p53-mediated transcription in the context of DNA or chromatin. This defect is specific for DNA molecules because inactive mutants fail to bind a consensus p53 response element in both free DNA and nucleosomes. Chromatin immunoprecipitation assays further substantiate the importance of the p53 C-terminal domain for the targeted localization of p53 and the concomitant recruitment of p300 onto p53-responsive genes. Moreover, a synthetic peptide comprising the last 30 amino acids of p53 interacts with the N-terminal and C-terminal domains of p53 and antagonizes p53-dependent transcription. Taken together, our data reveal a functional requirement for the p53 C-terminal domain in p53 transactivation and support a working model in which the C-terminus serves as a positive regulator for N-terminal activation and central DNA binding domains.

Vpr-binding protein antagonizes p53-mediated transcription via direct interaction with H3 tail.

HIV-1 Vpr-binding protein (VprBP) has been implicated in the regulation of both DNA replication and cell cycle progression, but its precise role remains unclear. Here we report that VprBP regulates the p53-induced transcription and apoptotic pathway. VprBP is recruited to p53-responsive promoters and suppresses p53 transactivation in the absence of stress stimuli. To maintain target promoters in an inactive state, VprBP stably binds to nucleosomes by recognizing unacetylated H3 tails. Promoter-localized deacetylation of H3 tails is a prerequisite for VprBP to tether and act as a bona fide inhibitor at p53 target genes. VprBP knockdown leads to activation of p53 target genes and causes an increase in DNA damage-induced apoptosis. Moreover, phosphorylation of VprBP at serine 895 impairs the ability of VprBP to bind H3 tails and to repress p53 transactivation. Our results thus reveal a new role for VprBP in regulation of the p53 signaling pathway, as well as molecular mechanisms of cancer development related to VprBP misregulation.

Selective requirement of H2B N-Terminal tail for p14ARF-induced chromatin silencing.

The N-terminal tail of histone H2B is believed to be involved in gene silencing, but how it exerts its function remains elusive. Here, we report the biochemical characterization of p14ARF tumor suppressor as a transcriptional repressor that selectively recognizes the unacetylated H2B tails on nucleosomes. The p14ARF-H2B tail interaction is functional, as the antagonistic effect of p14ARF on chromatin transcription is lost upon deletion or acetylation of H2B tails. Gene expression profiling and chromatin immunoprecipitation studies emphasize the significance of H2B deacetylation and p14ARF recruitment in establishing a repressive environment over the cell cycle regulatory genes. Moreover, HDAC1-mediated H2B deacetylation, especially at K20, constitutes an essential step in tethering p14ARF near target promoters. Our results thus reveal a hitherto unknown role of p14ARF in the regulation of chromatin transcription, as well as molecular mechanisms governing the repressive action of p14ARF.

Histone variant H3.3 stimulates HSP70 transcription through cooperation with HP1γ.

Histone variant H3.3 and heterochromatin protein 1γ (HP1γ) are two functional components of chromatin with role in gene transcription. However, the regulations of their dynamics during transcriptional activation and the molecular mechanisms underlying their actions remain poorly understood. Here, we provide evidence that heat shock-induced transcription of the human HSP70 gene is regulated via the coordinated and interdependent action of H3.3 and HP1γ. H3.3 and HP1γ are rapidly co-enriched at the human HSP70 promoters upon heat shock in a manner that closely parallels the initiation of transcription. Knockdown of H3.3 prevents the stable recruitment of HP1γ, inhibits active histone modifications, and attenuates HSP70 promoter activity. Likewise, knockdown of HP1γ leads to the decreased levels of H3.3 in the promoter regions and the repression of HSP70 genes. HP1γ selectively recognizes particular modification states of H3.3 in the nucleosome for its action. Moreover, HP1γ is overexpressed in three representative cancer cell lines, and its knockdown leads to reduction in HSP70 gene transcription and inhibition of cancer cell proliferation. We conclude that the physical and functional interactions between H3.3 and HP1γ make a unique contribution to acute HSP70 transcription and cancer development related to the misregulation of this transcription event.

Cooperative action of TIP48 and TIP49 in H2A.Z exchange catalyzed by acetylation of nucleosomal H2A.

H2A.Z is an evolutionarily conserved H2A variant that plays a key role in the regulation of chromatin transcription. To understand the molecular mechanism of H2A.Z exchange, we purified two distinct H2A.Z-interacting complexes termed the small and big complexes from a human cell line. The big complex contains most components of the SRCAP chromatin remodeling and TIP60 HAT complexes, whereas the small complex possesses only a subset of SRCAP and TIP60 subunits. Our exchange analysis revealed that both small and big complexes enhance the incorporation of H2A.Z-H2B dimer into the nucleosome. In addition, TIP60-mediated acetylation of nucleosomal H2A specifically facilitates the action of the small complex in the H2A.Z exchange reaction. Among factors present in the small complex, we determined that TIP48 and TIP49 play a major role in catalyzing H2A acetylation-induced H2A.Z exchange via their ATPase activities. Overall, our work uncovers the previously-unrecognized role of TIP48 and TIP49 in H2A.Z exchange and a novel epigenetic mechanism controlling this process.

Requirement of histone methyltransferase SMYD3 for estrogen receptor-mediated transcription.

SMYD3 is a SET domain-containing protein with histone methyltransferase activity on histone H3-K4. Recent studies showed that SMYD3 is frequently overexpressed in different types of cancer cells, but how SMYD3 regulates the development and progression of these malignancies remains unknown. Here, we report the previously unrecognized role of SMYD3 in estrogen receptor (ER)-mediated transcription via its histone methyltransferase activity. We demonstrate that SMYD3 functions as a coactivator of ERalpha and potentiates ERalpha activity in response to ligand. SMYD3 directly interacts with the ligand binding domain of ER and is recruited to the proximal promoter regions of ER target genes upon gene induction. Importantly, our chromatin immunoprecipitation analyses provide compelling evidence that SMYD3 is responsible for the accumulation of di- and trimethylation of H3-K4 at the induced ER target genes. Furthermore, RNA interference-directed down-regulation of SMYD3 reveals that SMYD3 is required for ER-regulated gene transcription in estrogen signaling pathway. Thus, our results identify SMYD3 as a new coactivator for ER-mediated transcription, providing a possible link between SMYD3 overexpression and breast cancer.

FACT-mediated exchange of histone variant H2AX regulated by phosphorylation of H2AX and ADP-ribosylation of Spt16.

The phosphorylation of histone variant H2AX at DNA double-strand breaks is believed to be critical for recognition and repair of DNA damage. However, little is known about the molecular mechanism regulating the exchange of variant H2AX with conventional H2A in the context of the nucleosome. Here, we isolate the H2AX-associated factors, which include FACT (Spt16/SSRP1), DNA-PK, and PARP1 from a human cell line. Our analyses demonstrate that the H2AX-associated factors efficiently promote both integration and dissociation of H2AX and this exchange reaction is mainly catalyzed by FACT among the purified factors. The phosphorylation of H2AX by DNA-PK facilitates the exchange of nucleosomal H2AX by inducing conformational changes of the nucleosome. In contrast, poly-ADP-ribosylation of Spt16 by PARP1 significantly inhibits FACT activities for H2AX exchange. Thus, these data establish FACT as the major regulator involved in H2AX exchange process that is modulated by H2AX phosphorylation and Spt16 ADP-ribosylation.

Isolation and characterization of a novel H1.2 complex that acts as a repressor of p53-mediated transcription.

Linker histone H1 has been generally viewed as a global repressor of transcription by preventing the access of transcription factors to sites in chromatin. However, recent studies suggest that H1 can interact with other regulatory factors for its action as a negative modulator of specific genes. To investigate these aspects, we established a human cell line expressing H1.2, one of the H1 subtypes, for the purification of H1-interacting proteins. Our results showed that H1.2 can stably associate with sets of cofactors and ribosomal proteins that can significantly repress p53-dependent, p300-mediated chromatin transcription. This repressive action of H1.2 complex involves direct interaction of H1.2 with p53, which in turn blocks p300-mediated acetylation of chromatin. YB1 and PURalpha, two factors present in the H1.2 complex, together with H1.2 can closely recapitulate the repressive action of the entire H1.2 complex in transcription. Chromatin immunoprecipitation and RNA interference analyses further confirmed that the recruitment of YB1, PURalpha, and H1.2 to the p53 target gene Bax is required for repression of p53-induced transcription. Therefore, these results reveal a previously unrecognized function of H1 as a transcriptional repressor as well as the underlying mechanism involving specific sets of factors in this repression process.

Transforming growth factor-beta1 represses E-cadherin production via slug expression in lens epithelial cells.

PURPOSE: TGFbeta is a potent candidate for epithelial-mesenchymal transition (EMT) during the development of anterior polar cataracts in the human lens. The Snail superfamily is involved in EMT through the repression of E-cadherin production. This study was conducted to determine whether the Snail gene family is activated in the process of TGFbeta1-induced EMT and how TGFbeta1 regulates the expression of this gene family. METHODS: Total RNA extracted from human cataract samples was subjected to the real-time PCR quantification of Slug mRNA. Induction of Slug expression by TGFbeta1 (10 ng/mL) in lens epithelial cells was determined by RT-PCR, immunostaining, immunoblot analysis, and Slug promoter analysis. A series of Slug promoter deletion constructs was used to identify the putative regulatory element responsive to TGF signaling. Chromatin immunoprecipitation was performed to determine whether Sp1 associates with the endogenous Slug promoter. Inhibition of Slug expression with Slug siRNA was used to investigate the role of Slug in TGFbeta-mediated EMT. RESULTS: Slug levels were highly upregulated in lens epithelial cells obtained from patients with anterior polar cataracts. Treatment of TGFbeta1 induced the expression of Slug in both lens and other epithelial cells in vitro. TGFbeta1-induced Slug expression was significantly inhibited by the MEK- and JNK/SAPK-specific inhibitors, but not by transfection with dominant-negative forms of Smads or small GTPase proteins, indicating that MAPK pathways are involved in the regulation of Slug expression by TGFbeta1. The Slug promoter analysis revealed that the Sp1 binding site in the Slug promoter is responsible for TGFbeta1-induced Slug expression. In addition, the TGFbeta1-mediated repression of E-cadherin was significantly inhibited by Slug siRNA. CONCLUSIONS: These data suggest that TGFbeta1 induces Slug expression and that the repression of E-cadherin production by TGFbeta1 is mediated by the induction of Slug in lens epithelial cells.

Adenomatous polyposis coli is down-regulated by the ubiquitin-proteasome pathway in a process facilitated by Axin.

Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of beta-catenin, a key effector of Wnt signaling. Truncation mutations in APC are responsible for familial and sporadic colorectal tumors due to failure in the down-regulation of beta-catenin. While the regulation of beta-catenin by APC has been extensively studied, the regulation of APC itself has received little attention. Here we show that the level of APC is down-regulated by the ubiquitin-proteasome pathway and that Wnt signaling inhibits the process. The domain responsible for the down-regulation and direct ubiquitination was identified. We also show an unexpected role for Axin in facilitating the ubiquitination-proteasome-mediated down-regulation of APC through the oligomerization of Axin. Our results suggest a new mechanism for the regulation of APC by Axin and Wnt signaling.

Hepatocyte growth factor induces proliferation of lens epithelial cells through activation of ERK1/2 and JNK/SAPK.

PURPOSE: Posterior capsule opacification (PCO) is caused by proliferation and migration of lens epithelial cells (LECs) remaining after cataract surgery. In this study, the effect of HGF in LECs and the signaling pathways that contribute to HGF-induced proliferation were investigated. METHODS: Capsular bags prepared from porcine eyes were maintained in serum-free DMEM. The human lens epithelial B3 cells (HLE B3) and rat lens epithelial explants were cultured in MEM supplemented with 20% FCS and medium 199 with 0.1% BSA, respectively. Cell proliferation was determined by MTT assay, proliferating cell nuclear antigen (PCNA) expression, or flow cytometry. An antisense oligonucleotide was used to inhibit cyclin D1 expression. Activation of the mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways was detected by immunoblot analysis. RESULTS: The proliferation of LECs in a capsular bag culture was significantly inhibited by treatment with the neutralizing antibody for HGF receptor. Stimulation of HLE B3 with hepatocyte growth factor (HGF) activated the MAPKs, ERK, and JNK/SAPK, but not p38. Activation of both ERK and JNK/SAPK was necessary for the HGF-stimulated induction of cyclin D1, which in turn was necessary for the HGF-induced proliferation of LECs. PI3K also participated in the regulation of cyclin D1 expression upstream of ERK and JNK/SAPK. CONCLUSIONS: The data indicate that HGF is a potent growth factor for LECs and may contribute to the development of PCO and suggest that the signaling pathways involved in HGF-stimulated proliferation may constitute potential therapeutic targets in the treatment of PCO.