Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

Genetic and genomic studies of pathogenic EXOSC2 mutations in the newly described disease SHRF implicate the autophagy pathway in disease pathogenesis.

Missense mutations in the RNA exosome component exosome component 2 (EXOSC2), also known as ribosomal RNA-processing protein 4 (RRP4), were recently identified in two unrelated families with a novel syndrome known as Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies (SHRF, #OMIM 617763). Little is known about the mechanism of the SHRF pathogenesis. Here we have studied the effect of mutations in EXOSC2/RRP4 in patient-derived lymphoblasts, clustered regularly interspaced short palindromic repeats (CRISPR)-generated mutant fetal keratinocytes and Drosophila. We determined that human EXOSC2 is an essential gene and that the pathogenic G198D mutation prevents binding to other RNA exosome components, resulting in protein and complex instability and altered expression and/or activities of critical genes, including those in the autophagy pathway. In parallel, we generated multiple CRISPR knockouts of the fly rrp4 gene. Using these flies, as well as rrp4 mutants with Piggy Bac (PBac) transposon insertion in the 3'UTR and RNAi flies, we determined that fly rrp4 was also essential, that fly rrp4 phenotypes could be rescued by wild-type human EXOSC2 but not the pathogenic form and that fly rrp4 is critical for eye development and maintenance, muscle ultrastructure and wing vein development. We found that overexpression of the transcription factor MITF was sufficient to rescue the small eye and adult lethal phenotypes caused by rrp4 inhibition. The autophagy genes ATG1 and ATG17, which are regulated by MITF, had similar effect. Pharmacological stimulation of autophagy with rapamycin also rescued the lethality caused by rrp4 inactivation. Our results implicate defective autophagy in SHRF pathogenesis and suggest therapeutic strategies.

ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes.

ZNF750 controls epithelial homeostasis by inhibiting progenitor genes while inducing differentiation genes, a role underscored by pathogenic ZNF750 mutations in cancer and psoriasis. How ZNF750 accomplishes these dual gene regulatory impacts is unknown. Here, we characterized ZNF750 as a transcription factor that binds both the progenitor and differentiation genes that it controls at a CCNNAGGC DNA motif. ZNF750 interacts with the pluripotency transcription factor KLF4 and chromatin regulators RCOR1, KDM1A, and CTBP1/2 through conserved PLNLS sequences. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) and gene depletion revealed that KLF4 colocalizes ∼ 10 base pairs from ZNF750 at differentiation target genes to facilitate their activation but is unnecessary for ZNF750-mediated progenitor gene repression. In contrast, KDM1A colocalizes with ZNF750 at progenitor genes and facilitates their repression but is unnecessary for ZNF750-driven differentiation. ZNF750 thus controls differentiation in concert with RCOR1 and CTBP1/2 by acting with either KDM1A to repress progenitor genes or KLF4 to induce differentiation genes.

[Effect of carnosol against proliferative activity of breast cancer cells and its estrogen receptor subtype's mediation and regulation mechanisms].

Carnosol has been proved to have anti-breast cancer effect in previous research. But its ER subtype's specific regulation and mediation mechanisms remain unclear. The aim of this study is to observe the effect of carnosol on cell proliferation and its estrogen receptor α and ß's specific regulation and mediation mechanisms with ER positive breast cancer T47D cell. With estrogen receptor α and ß antagonists MPP and PHTPP as tools, the MTT cell proliferation assay was performed to observe the effect of carnosol on T47D cell proliferation. The changes in the T47D cell proliferation cycle were detected by flow cytometry. The effect of carnosol on ERα and ERß expressions of T47D cells was measured by Western blot. The findings showed that 1 x 10(-5)-1 x 10(-7) mol x L(-1) carnosol could significantly inhibit the T47D cell proliferation, which could be enhanced by MPP or weakened by PHTPP. Meanwhile, 1 x 10(-5) mol x L(-1) or 1 x 10(-6) mol x L(-1) carnosol could significantly increase ERα and ERß expressions of T47D cells, and remarkably increase ERα/ERß ratio. The results showed that carnosol showed the inhibitory effect on the proliferation of ER positive breast cancer cells through target cell ER, especially ERß pathway. In the meantime, carnosol could regulate expressions and proportions of target cell ER subtype ERα and ERß.

CDK4 coexpression with Ras generates malignant human epidermal tumorigenesis.

Ras acts with other proteins to induce neoplasia. By itself, however, strong Ras signaling can suppress proliferation of normal cells. In primary epidermal cells, we found that oncogenic Ras transiently decreases cyclin-dependent kinase (CDK) 4 expression in association with cell cycle arrest in G1 phase. CDK4 co-expression circumvents Ras growth suppression and induces invasive human neoplasia resembling squamous cell carcinoma. Tumorigenesis is dependent on CDK4 kinase function, with cyclin D1 required but not sufficient for this process. In facilitating escape from G1 growth restraints, Ras and CDK4 alter the composition of cyclin D and cyclin E complexes and promote resistance to growth inhibition by INK4 cyclin-dependent kinase inhibitors. These data identify a new role for oncogenic Ras in CDK4 regulation and highlight the functional importance of CDK4 suppression in preventing uncontrolled growth.

CDK4 regulation by TNFR1 and JNK is required for NF-kappaB-mediated epidermal growth control.

Nuclear factor kappaB (NF-kappaB) mediates homeostatic growth inhibition in the epidermis, and a loss of NF-kappaB function promotes proliferation and oncogenesis. To identify mechanisms responsible for these effects, we impaired NF-kappaB action in the epidermis by three different genetic approaches, including conditional NF-kappaB blockade. In each case, epidermal hyperplasia was accompanied by an increase in both protein levels and tissue distribution of the G1 cell cycle kinase, CDK4. CDK4 up-regulation required intact TNFR1 and c-Jun NH2-terminal kinase (JNK) function. Cdk4 gene deletion concomitant with conditional NF-kappaB blockade demonstrated that CDK4 is required for growth deregulation. Therefore, epidermal homeostasis depends on antagonist regulation of CDK4 expression by NF-kappaB and TNFR1/JNK.

Tumor necrosis factor receptor 1/c-Jun-NH2-kinase signaling promotes human neoplasia.

The tumor necrosis factor alpha receptor (TNFR1) activates downstream effectors that include the mitogen-activated protein kinase kinase 7 (MKK7)/c-Jun-NH(2)-kinase (JNK)/activator protein 1 (AP1) cascade. Here, we report that JNK is activated in a majority of spontaneous human squamous cell carcinomas (SCC). JNK pathway induction bypassed cell cycle restraints induced by oncogenic Ras and cooperated with Ras to convert normal human epidermis into tumors indistinguishable from SCC, confirming its oncogenic potency in human tissue. Inhibiting MKK7, JNK, and AP1 as well as TNFR1 itself using genetic, pharmacologic, or antibody-mediated approaches abolished invasive human epidermal neoplasia in a tumor cell autonomous fashion. The TNFR1/MKK7/JNK/AP1 cascade thus promotes human neoplasia and represents a potential therapeutic target for human epithelial cancers.

ER-mediated anti-tumor effects of shikonin on breast cancer.

Estrogen receptor (ER) is expressed in most Breast cancer (BC) patients. G protein-coupled estrogen receptor (GPER), which is a membrane-bound estrogen receptor, is associated with the tumor development and progression in BC. Shikonin (SK) is a natural compound that is known to have anti-tumor effects. This study aims to assess the effects of shikonin on the cell proliferation, cell cycle and cell apoptosis of BC and whether the effects are related to ER/GPER signaling pathway. The results demonstrated that shikonin inhibited the cellular proliferation of MCF-7 BC cells via G0/G1 arrest and apoptosis in concentration-dependent manner. The anti-proliferative effect of SK on SK-BR-3 BC cells was associated with apoptosis. Both ERα and GPER were expressed in MCF-7 cells, while ERα were negative and GPER were positive in SK-BR-3 cells. Furthermore, shikonin downregulated the expression of ERα and GPER, and this effect was not affected by the estrogen environment. In addition, shikonin downregulated the EGFR and p-ERK expression in MCF-7 and SK-BR-3, which was also not affected by the estrogen environment. EGFR and p-ERK were still suppressed by co-treatment with the selective GPER against G1 or antagonist G15. In conclusion, these results suggest that shikonin shows anti-tumor effects on MCF-7 and SK-BR-3 cells. The effects seem to be associated with EGFR/p-ERK downregulation via ERα and GPER inhibition.

CSNK1a1 Regulates PRMT1 to Maintain the Progenitor State in Self-Renewing Somatic Tissue.

Somatic progenitors sustain tissue self-renewal while suppressing premature differentiation. Protein arginine methyltransferases (PRMTs) affect many processes; however, their role in progenitor function is incompletely understood. PRMT1 was found to be the most highly expressed PRMT in epidermal progenitors and the most downregulated PRMT during differentiation. In targeted mouse knockouts and in long-term regenerated human mosaic epidermis in vivo, epidermal PRMT1 loss abolished progenitor self-renewal and led to premature differentiation. Mass spectrometry of the PRMT1 protein interactome identified the CSNK1a1 kinase, which also proved essential for progenitor maintenance. CSNK1a1 directly bound and phosphorylated PRMT1 to control its genomic targeting to PRMT1-sustained proliferation genes as well as PRMT1-suppressed differentiation genes. Among the latter were GRHL3, whose derepression was required for the premature differentiation seen with PRMT1 and CSNK1a1 loss. Maintenance of the progenitors thus requires cooperation by PRMT1 and CSNK1a1 to sustain proliferation gene expression and suppress premature differentiation driven by GRHL3.

A LncRNA-MAF:MAFB transcription factor network regulates epidermal differentiation.

Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.

Comparison between the efficacies of curcumin and puerarin in C57BL/6 mice with steatohepatitis induced by a methionine- and choline-deficient diet.

Non-alcoholic fatty liver disease (NAFLD) is a prevalent disease, which features an abnormal accumulation of lipids inside hepatocytes. Steatohepatitis plays a critical role in the process resulting in liver fibrosis and cirrhosis. Curcumin and puerarin are herbal products widely used in Asia, which are believed to have therapeutic benefits for alleviating the symptoms of steatohepatitis. In this study, mice models of steatohepatitis induced by a methionine- and choline-deficient diet (MCD) were established to compare the pharmacological actions of curcumin and puerarin. The results showed that curcumin and puerarin exerted inhibitory effects against MCD-induced steatohepatitis in mice. Briefly, curcumin and puerarin significantly downregulated the levels of tumor necrosis factor-α in the blood serum of mice (P<0.01, versus the MCD group). In addition, the levels of triglycerides, total cholesterol and low density lipoproteins in the serum were significantly reduced by puerarin treatment (P<0.05, versus the MCD group). The concentration of interleukin-6 was downregulated by curcumin only (P<0.01, versus the MCD group). Curcumin and puerarin significantly increased the levels of peroxisome proliferator-activated receptor-γ (PPARγ; P<0.05, versus the MCD group). Moreover, increased nuclear factor-κB (NF-κB) was markedly attenuated by curcumin (P<0.05, versus the MCD group). In conclusion, curcumin and puerarin appear to exert different actions against steatohepatitis. It is possible that puerarin regulated lipid metabolism in the 'first hit' stage through the PPARγ pathway, while curcumin inhibited the inflammatory response in the 'second hit' stage through the NF-κB pathway.

ACTL6a enforces the epidermal progenitor state by suppressing SWI/SNF-dependent induction of KLF4.

Somatic progenitors suppress differentiation to maintain tissue self-renewal. The mammalian SWI/SNF chromatin-remodeling complex regulates nucleosome packaging to control differentiation in embryonic and adult stem cells. Catalytic Brg1 and Brm subunits are required for these processes; however, the roles of SWI/SNF regulatory subunits are not fully understood. Here, we show that ACTL6a/BAF53A modulates the SWI/SNF complex to suppress differentiation in epidermis. Conditional loss of ACTL6a resulted in terminal differentiation, cell-cycle exit, and hypoplasia, whereas ectopic expression of ACTL6a promoted the progenitor state. A significant portion of genes regulated by ACTL6a were found to also be targets of KLF4, a known activator of epidermal differentiation. Mechanistically, we show that ACTL6a prevents SWI/SNF complex binding to promoters of KLF4 and other differentiation genes and that SWI/SNF catalytic subunits are required for full induction of KLF4 targets. Thus, ACTL6a controls the epidermal progenitor state by sequestering SWI/SNF to prevent activation of differentiation programs.

The c-Jun NH2-terminal kinase 2 plays a dominant role in human epidermal neoplasia.

The c-Jun NH(2)-terminal kinase (JNK) signaling cascade has been implicated in a wide range of diseases, including cancer. It is unclear how different JNK proteins contribute to human cancer. Here, we report that JNK2 is activated in more than 70% of human squamous cell carcinoma (SCC) samples and that inhibition of JNK2 pharmacologically or genetically impairs tumorigenesis of human SCC cells. Most importantly, JNK2, but not JNK1, is sufficient to couple with oncogenic Ras to transform primary human epidermal cells into malignancy with features of SCC. JNK2 prevents Ras-induced cell senescence and growth arrest by reducing the expression levels of the cell cycle inhibitor p16 and the activation of NF-kappaB. On the other hand, JNK, along with phosphoinositide 3-kinase, is essential for Ras-induced glycolysis, an energy-producing process known to benefit cancer growth. These data indicate that JNK2 collaborates with other oncogenes, such as Ras, at multiple molecular levels to promote tumorigenesis and hence represents a promising therapeutic target for cancer.

Evidence of EGR1 as a differentially expressed gene among proliferative skin diseases.

Hyperproliferative epidermal disorders range from benign hyperplasias such as psoriasis to basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), the two most common cancers in the US. While they all arise from the epidermis, these diseases differ dramatically in biological behavior and their underlying gene expression patterns have not been compared. We thus examined mRNA transcript levels in these disorders to identify and further characterize differentially expressed genes. Transcript expression patterns distinguish these disorders and identify EGR1, among other genes, whose epidermal expression is decreased in BCC and SCC but is elevated in psoriasis. Egr-1 inhibits growth of benign and malignant epidermal cells in vitro and appears to suppress both Cdc25A expression and Cdk2 dephosphorylation. These data indicate that gene expression profiling can differentiate epidermal hyperproliferative diseases and suggest that Egr-1 may play a role in preventing uncontrolled epidermal growth.

NF-kappaB RelA opposes epidermal proliferation driven by TNFR1 and JNK.

NF-kappaB inhibition promotes epidermal tumorigenesis; however, whether this reflects an underlying role in homeostasis or a special case confined to neoplasia is unknown. Embryonic lethality of mice lacking NF-kappaB RelA has hindered efforts to address this. We therefore generated developmentally mature RelA(-/-) skin. RelA(-/-) epidermis displays hyperplasia without abnormal differentiation, inflammation, or apoptosis. Hyperproliferation is TNFR1-dependent because Tnfr1 deletion normalized cell division. TNFR1-dependent JNK activation occurred in RelA(-/-) epidermis, and JNK inhibition abolished hyperproliferation due to RelA deficiency. Thus, RelA antagonizes TNFR1-JNK proliferative signals in epidermis and plays a nonredundant role in restraining epidermal growth.

NF-kappaB blockade and oncogenic Ras trigger invasive human epidermal neoplasia.

The nuclear factor NF-kappaB and oncogenic Ras can alter proliferation in epidermis, the most common site of human cancer. These proteins are implicated in epidermal squamous cell carcinoma in mice, however, the potential effects of altering their function are uncertain. Whereas inhibition of NF-kappaB enhances apoptosis in certain tumours, blockade of NF-kappaB predisposes murine skin to squamous cell carcinoma. Because therapeutics inhibiting Ras and NF-kappaB pathways are being developed to treat human cancer, it is essential to assess the effects of altering these regulators. The medical relevance of murine studies is limited, however, by differences between mouse and human skin, and by the greater ease of transforming murine cells. Here we show that in normal human epidermal cells both NF-kappaB and oncogenic Ras trigger cell-cycle arrest. Growth arrest triggered by oncogenic Ras can be bypassed by IkappaBalpha-mediated blockade of NF-kappaB, generating malignant human epidermal tissue resembling squamous cell carcinoma. Human cell tumorigenesis is dependent on laminin 5 and alpha6beta4 integrin. Thus, IkappaBalpha circumvents restraints on growth promotion induced by oncogenic Ras and can act with Ras to induce invasive human tissue neoplasia.