SummaryAuxin response factors (ARFs) bind auxin response promoter elements and mediate transcriptional responses to auxin. Five of the 22 ARF genes in Arabidopsis thaliana encode ARFs with glutamine-rich middle domains. Four of these can activate transcription and have been ascribed developmental functions. We show that ARF19, the fifth Q-rich ARF, also activates transcription. Mutations in ARF19 have little effect on their own, but in combination with mutations in NPH4/ARF7, encoding the most closely related ARF, they cause several phenotypes including a drastic decrease in lateral and adventitious root formation and a decrease in leaf cell expansion. These results indicate that auxin induces lateral roots and leaf expansion by activating NPH4/ARF7 and ARF19. Auxin induces the ARF19 gene, and NPH4/ARF7 and ARF19 together are required for expression of one of the arf19 mutant alleles, suggesting that a positive feedback loop regulates leaf expansion and/or lateral root induction.

IntroductionPhiladelphia chromosome-negative myeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic disorders that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). 1,2 Recent studies have confirmed the pathogenetic involvement of an acquired, somatic, gain-offunction, activating, point mutation JAK2V617F in MPNs. [3][4][5][6] This represents a guanine to thymidine mutation in exon 14 resulting in a valine to phenylalanine substitution at codon 617 in the JH2 or pseudokinase domain of the JAK2 gene (a member of the Janus kinase [JAK] family of nonreceptor tyrosine kinases, JAK1, JAK2, JAK3, and TYK2). 2,6 Highly sensitive assays for JAK2 have determined that the JAK2V617F mutation is present in 90% of patients with PV and approximately 50% to 60% of patients with ET or PMF. 7 In addition, a subset of patients, most commonly with PV, are homozygous for the JAK2V617F allele, the result of copy-neutral loss of heterozygosity at the JAK2 locus, especially in patients with PV. 2,7,8 Mutations in exon 12 of JAK2 are present in almost all patients with PV who are JAK2V617F negative. 9,10 The JAK proteins function in the cytoplasm to relay signals initiated by membrane-bound cytokine receptors. Engagement of the receptor results in the phosphorylation of the receptor and JAK2, which recruits its substrate proteins such as signal transducers and activators of transcription (STATs). 11,12 STATs, especially STAT3 and STAT5, translocate to the nucleus and transactivate many genes involved in cell proliferation and survival (eg, Bcl-xL, cyclin D1, and PIM1). 8,11,12 The V617F mutation in JAK2 also activates the downstream signaling pathways through the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK). This contributes to diminished apoptosis of the hematopoietic progenitor cells (HPCs). 2,8 Overexpression of JAK2V617F in murine Ba/F3 cells with coexpression of the erythropoietin receptor (EpoR) confers in vitro cytokine-independent growth. 3,13 Recently, it was shown that enforced expression of JAK2V617F in human hematopoietic stem cells and myeloid progenitors directed differentiation toward the erythroid lineage, along with increased expression and phosphorylation of GATA-1 and decreased expression of PU.1. 14-16 JAK2V617F expression in retroviral models and in transgenic mice is sufficient to cause myeloproliferative disorders in the mice that recapitulate many clinicopathologic features observed in human PV, ET, and PMF. [17][18][19][20][21] 22,23 In vivo studies in mouse models have also shown that mutant JAK2V617F represents a novel target for therapeutic intervention with JAK2-selective tyrosine kinase inhibitors in MPNs. 21,24 For example, TG101348 inhibits myeloproliferation and myelofibrosis in a murine model of JAK2V617F-induced polycythemia. 21,22 Early clinical trials of several JAK2-selective kinase inhibitors (eg, XL019, TG101348, and INCB18424) are under way in JAK2-driven MPNs with poor prognosis (eg, PMF). ...

The PRC2 complex protein EZH2 is a histone methyltransferase that is known to bind and recruit DNMT1 to the DNA to modulate DNA methylation. Here, we determined that the pan-HDAC inhibitor panobinostat (LBH589) treatment depletes DNMT1 and EZH2 protein levels, disrupts the interaction of DNMT1 with EZH2, as well as de-represses JunB in human acute leukemia cells. Similar to treatment with the hsp90 inhibitor 17-DMAG, treatment with panobinostat also inhibited the chaperone association of heat shock protein 90 with DNMT1 and EZH2, which promoted the proteasomal degradation of DNMT1 and EZH2. Unlike treatment with the DNA methyltransferase inhibitor decitabine, which demethylates JunB promoter DNA, panobinostat treatment mediated chromatin alterations in the JunB promoter. Combined treatment with panobinostat and decitabine caused greater attenuation of DNMT1 and EZH2 levels than either agent alone, which was accompanied by more JunB de-repression and loss of clonogenic survival of K562 cells. Co-treatment with panobinostat and decitabine also caused more loss of viability of primary AML but not normal CD34+ bone marrow progenitor cells. Collectively, these findings indicate that co-treatment with panobinostat and decitabine targets multiple epigenetic mechanisms to de-repress JunB and exerts antileukemia activity against human acute myeloid leukemia cells.

New therapies toward heart and blood vessel disorders may emerge from the development of Hsp90 inhibitors. Several independent studies suggest potent anti-inflammatory activities of those agents in human tissues. The molecular mechanisms responsible for their protective effects in the vasculature remain unclear. The present study demonstrates that the transcription factor p53, an Hsp90 client protein, is crucial for the maintenance of vascular integrity, protects again LPS-induced endothelial barrier dysfunction, and is involved in the mediation of the anti-inflammatory activity of Hsp90 inhibitors in lung tissues. p53 silencing by siRNA decreased transendothelial resistance (a measure of endothelial barrier function). A similar effect was induced by the p53 inhibitor pifithrin, which also potentiated the LPS-induced hyperpermeability in human lung microvascular endothelial cells (HLMVEC). On the other hand, p53 induction by nutlin suppressed the LPS-induced vascular barrier dysfunction. LPS decreased p53 expression in lung tissues and that effect was blocked by pretreatment with Hsp90 inhibitors both in vivo and in vitro. Furthermore, the Hsp90 inhibitor 17-allyl-amino-demethoxy-geldanamycin suppressed the LPS-induced overexpression of the p53 negative regulator MDMX as well as p53 and MDM2 (another p53 negative regulator) phosphorylation in HLMVEC. Both negative p53 regulators were downregulated by LPS in vivo. Chemically induced p53 overexpression resulted in the suppression of LPS-induced RhoA activation and MLC2 phosphorylation, whereas p53 suppression caused the opposite effects. These observations reveal new mechanisms for the anti-inflammatory actions of Hsp90 inhibitors, i.e., the induction of the transcription factor p53, which in turn can orchestrate robust vascular anti-inflammatory responses both in vivo and in vitro.

Increased levels of misfolded polypeptides in the endoplasmic reticulum (ER) triggers the dissociation of glucose-regulated protein 78 (GRP78) from the three transmembrane ER-stress mediators, i.e., protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF6), and inositol-requiring enzyme 1α, which results in the adaptive unfolded protein response (UPR). In the present studies, we determined that histone deacetylase-6 (HDAC6) binds and deacetylates GRP78. Following treatment with the pan-histone deacetylase inhibitor panobinostat (Novartis Pharmaceuticals), or knockdown of HDAC6 by short hairpin RNA, GRP78 is acetylated in 11 lysine residues, which dissociates GRP78 from PERK. This is associated with the activation of a lethal UPR in human breast cancer cells. Coimmunoprecipitation studies showed that binding of HDAC6 to GRP78 requires the second catalytic and COOH-terminal BUZ domains of HDAC6. Treatment with panobinostat increased the levels of phosphorylated-eukaryotic translation initiation factor (p-eIF2α), ATF4, and CAAT/enhancer binding protein homologous protein (CHOP). Panobinostat treatment also increased the proapoptotic BIK, BIM, BAX, and BAK levels, as well as increased the activity of caspase-7. Knockdown of GRP78 sensitized MCF-7 cells to bortezomib and panobinostat-induced UPR and cell death. These findings indicate that enforced acetylation and decreased binding of GRP78 to PERK is mechanistically linked to panobinostat-induced UPR and cell death of breast cancer cells. Mol Cancer Ther; 9(4); 942-52. ©2010 AACR.