Thippeswamy Gulappa

A Novel Small-Molecule Inhibitor of Mcl-1 Blocks Pancreatic Cancer Growth In Vitro and In Vivo

Using a high-throughput screening (HTS) approach, we have identified and validated several small-molecule Mcl-1 inhibitors (SMI). Here, we describe a novel selective Mcl-1 SMI inhibitor, 2 (UMI-77), developed by structure-based chemical modifications of the lead compound 1 (UMI-59). We have characterized the binding of UMI-77 to Mcl-1 by using complementary biochemical, biophysical, and computational methods and determined its antitumor activity against a panel of pancreatic cancer cells and an in vivo xenograft model. UMI-77 binds to the BH3-binding groove of Mcl-1 with Ki of 490 nmol/L, showing selectivity over other members of the antiapoptotic Bcl-2 family. UMI-77 inhibits cell growth and induces apoptosis in pancreatic cancer cells in a time- and dose-dependent manner, accompanied by cytochrome c release and caspase-3 activation. Coimmunoprecipitation experiments revealed that UMI-77 blocks the heterodimerization of Mcl-1/Bax and Mcl-1/Bak in cells, thus antagonizing the Mcl-1 function. The Bax/Bak-dependent induction of apoptosis was further confirmed using murine embryonic fibroblasts that are Bax- and Bak-deficient. In an in vivo BxPC-3 xenograft model, UMI-77 effectively inhibited tumor growth. Western blot analysis in tumor remnants revealed enhancement of proapoptotic markers and significant decrease of survivin. Collectively, these promising findings show the therapeutic potential of Mcl-1 inhibitors against pancreatic cancer and warrant further preclinical investigations. Mol Cancer Ther; 13(3); 565–75. ©2013 AACR.

Molecular regulation of gonadotropin receptor expression: relationship to sterol metabolism.

We have identified a specific LHR mRNA binding protein that selectively binds to the polypyrimidine-rich bipartite sequence in the coding region of the LHR mRNA and accelerates its degradation. This process has been shown to be one of the mechanisms that is responsible for the loss of the steady-state levels of LHR mRNA following the preovulatory LH surge or the down regulation of the receptor in response to the administration of a pharmacological dose of LH or hCG. The trans factor, designated as the LHR mRNA binding protein (LRBP), was purified and its identity was established as being mevalonate kinase, an enzyme involved in cholesterol biosynthesis. When mevalonate kinase expression was abolished by treating cultured luteal cells with 25-hydroxycholesterol, the ability to undergo LH-induced down regulation of LHR mRNA was completely abrogated. Examination of the crystal structure of mevalonate kinase coupled with mutagenesis of the critical residues in the catalytic site revealed that the catalytic site is in close proximity to the LHR mRNA binding site. Further studies revealed that mevalonate kinase causes LHR mRNA degradation by acting as a translational suppressor by forming an untranslatable ribonucleoprotein (RNP) complex which is then targeted for degradation. These studies show that LHR expression in the ovary is regulated by a post-transcriptional mechanism mediated by mevalonate kinase thereby linking LHR expression with cholesterol metabolism.

Identification and characterization of proteins that selectively interact with the LHR mRNA binding protein (LRBP) in rat ovaries.

Luteinizing hormone receptor (LHR) mRNA binding protein (LRBP), identified as mevalonate kinase, has been shown to be a trans factor mediating the post-transcriptional regulation of LHR mRNA expression in ovaries. LRBP binds to the coding region of LHR mRNA and accelerates its degradation. Our previous studies in an in vitro system showed that LRBP represses the translation of LHR mRNA by forming an untranslatable ribonucleoprotein (mRNP) complex, further suggesting that the untranslatable mRNP complex is directed to the mRNA repression/decay machinery for subsequent mRNA turnover. In the present studies, we used yeast two-hybrid system to screen a cDNA library which was constructed from LHR down-regulated ovaries. Two proteins were identified interacting with LRBP: ribosomal protein S20 (RP S20) and ubiquitin conjugating enzyme 2i (UBCE2i). Their interactions with LRBP were confirmed by the mating assay, co-immunoprecipitation analyses and in vitro sumoylation assays. Furthermore, we show that LRBP is a target for modification by SUMO2/3 but not by SUMO1, at K256 and/or K345. Mutation of both lysine residues is sufficient to abrogate the sumoylation of LRBP. These findings suggest that the direct interaction of LRBP with the translation machinery, through RP S20, may be responsible for the transition of LHR mRNA to an untranslatable complex, and that sumoylation of LRBP may play a role in targeting the untranslatable mRNP complex to the mRNA decay machinery in specific cytoplasmic foci.

miR-122 Regulates LH Receptor Expression by Activating Sterol Response Element Binding Protein in Rat Ovaries

LH/human chorionic gonadotropin receptor (LHR) undergoes down-regulation during preovulatory LH surge or in response to exposure to a supraphysiological concentration of its ligands through a posttranscriptional mechanism involving an RNA binding protein designated as LHR mRNA binding protein (LRBP). miR-122, a short noncoding RNA, has been shown to mediate the up-regulation of LRBP. In the present study, we show that inhibition of miR-122 using a locked nucleic acid (LNA)-conjugated antagomir suppressed human chorionic gonadotropin (hCG)-induced up-regulation of LRBP as well as its association with LHR mRNA, as analyzed by RNA EMSA. Most importantly, inhibition of miR-122 resulted in the abolishment of hCG-mediated LHR mRNA down-regulation. We also show that the transcription factor, sterol regulatory element binding protein (SREBP) (SREBP-1a and SREBP-2 isoforms), is an intermediate in miR-122-mediated LHR mRNA regulation. HCG-stimulated increase in the activation of both SREBP-1a and SREBP-2 was inhibited by pretreatment with the miR-122 antagomir. The inhibition of cAMP/protein kinase A (PKA) and ERK pathways, upstream activators of miR-122, abolished SREBP activation after hCG treatment. SREBP-mediated regulation of LRBP expression is mediated by recruitment of LRBP promoter element to SREBP-1a, because chromatin immunoprecipitation assay revealed that association of LRBP promoter to SREBP was increased by hCG treatment. Pretreatment with miR-122 antagomir suppressed this response. Inhibition of SREBP activation by pretreating the rats with a chemical compound, fatostatin abrogated hCG-induced up-regulation of LRBP mRNA and protein. Fatostatin also inhibited LHR-LRBP mRNA-protein complex formation and LHR down-regulation. These results conclusively show that miR-122 plays a regulatory role in LH/hCG-induced LHR mRNA down-regulation by increasing LRBP expression through the activation of SREBP pathway.

The role of Rab5a GTPase in endocytosis and post-endocytic trafficking of the hCG-human luteinizing hormone receptor complex

This study examined the role of Rab5a GTPase in regulating hCG-induced internalization and trafficking of the hCG-LH receptor complex in transfected 293T cells. Coexpression of wild-type Rab5a (WT) or constitutively active Rab5a (Q79L) with LHR significantly increased hCG-induced LHR internalization. Conversely, coexpression of dominant negative Rab5a (S34N) with LHR reduced internalization. Confocal microscopy showed LHR colocalizing with Rab5a (WT) and Rab5a (Q79L) in punctuate structures. Coexpression of Rab5a (WT) and Rab5a (Q79L) with LHR significantly increased colocalization of LHR in early endosomes. Conversely, dominant negative Rab5a (S34N) decreased this colocalization. While Rab5a stimulated internalization of LHR, it significantly decreased LHR recycling to the cell surface and increased degradation. Dominant negative Rab5a (S34N) increased LHR recycling and decreased degradation. These results suggest that Rab5a plays a role in LHR trafficking by facilitating internalization and fusion to early endosomes, increasing the degradation of internalized receptor resulting in a reduction in LHR recycling.

Eukaryotic Initiation Factor 5A Plays an Essential Role in Luteinizing Hormone Receptor Regulation

Down-regulation of LH receptor (LHR) in the ovary by its ligand is mediated by a specific RNA-binding protein, designated LH receptor mRNA-binding protein (LRBP), through translational suppression and mRNA degradation. Using yeast 2-hybrid screens, we previously identified eukaryotic initiation factor 5A (eIF5A) as one of the proteins that interacts with LRBP during LHR mRNA down-regulation. The present study examined the role of eIF5A and its hypusination in the context of LHR mRNA down-regulation. The association of eIF5A with LRBP or LHR mRNA was determined using immunoprecipitation and RNA immunoprecipitation assays. The results showed that the association of eIF5A with the LHR mRNA-LRBP complex increased significantly during down-regulation. Furthermore, gel fractionation and the hypusination activity assay both showed increased hypusination of eIF5A during LHR mRNA down-regulation. Abolishment of hypusination by pretreatment with the chemical inhibitor GC7 prevented the association of eIF5A with LHR mRNA and LRBP. Inhibition of hypusination also reduced the extent of ligand-induced down-regulation of LHR mRNA as well as the expression of functional LHRs assessed by real-time PCR and (125)I-human chorionic gonadotropin (hCG) binding assays, respectively. The loss of human chorionic gonadotropin-mediated downstream signaling during LHR down-regulation was also restored by inhibition of hypusination of eIF5A. Thus, the present study, for the first time, reveals the crucial role of eIF5A and its hypusination in the regulation of LHR expression in the ovary.

Hypusination of eukaryotic initiation factor 5A via cAMP-PKA-ERK1/2 pathway is required for ligand-induced downregulation of LH receptor mRNA expression in the ovary

Luteinizing hormone receptor (LHR) mRNA expression in the ovary is regulated post-transcriptionally by an LH receptor mRNA binding protein (LRBP). Eukaryotic initiation factor 5A (EIF5A), identified as an LRBP-interacting protein plays a crucial role in LHR mRNA expression. In this study, we have demonstrated that during hCG-induced LHR downregulation, a significant upregulation of eIF5A mRNA expression and hypusination of eIF5A protein occurs in a time dependent manner. Pretreatment with H89, a specific inhibitor of PKA, and U0126, a specific inhibitor of ERK1/2 significantly inhibited both hCG-induced eIF5A mRNA expression and hypusination of eIF5A protein. Pretreatment with GC7, a specific inhibitor of eIF5A hypusination significantly abolished hCG-induced LRBP mRNA and protein expression. Furthermore, GC7 pretreatment significantly inhibited hCG-induced interaction of LRBP with LHR mRNA as assessed by RNA electrophoretic mobility gel shift assay (REMSA). GC7 treatment also reversed LHR mRNA downregulation. Taken together, these results suggest that hCG-induced LHR mRNA downregulation is mediated by cAMP-PKA-ERK1/2 signaling leading to activation of eIF5A hypusination.

Molecular regulation of LHCGR expression by miR-122 during follicle growth in the rat ovary

We have previously reported that LHCGR expression in the ovary is regulated through a post-transcriptional mechanism involving an mRNA binding protein designated as LRBP, which is regulated, at least in part, by a non-coding RNA, miR-122. Our present study examined the regulatory role of miR-122 in FSH-induced LHCGR expression during follicle development. Treatment of rat granulosa cells concurrently with FSH and 17β estradiol showed, as expected, a time-dependent increase in LHCGR mRNA levels as well as hCG-induced progesterone production. However, miR-122 expression was decreased during the early time periods, which preceded the increased expression of LHCGR mRNA. The role of miR-122 in FSH-induced LHCGR mRNA expression was then examined by overexpressing miR-122 prior to FSH stimulation by infecting granulosa cells with an adenoviral vector containing a miR-122 insert (AdmiR-122). Pretreatment with AdmiR-122 resulted in complete abrogation of FSH- mediated upregulation of LHCGR. AdmiR-122 also blocked FSH-induced decrease in LRBP expression and increased the binding of LHCGR mRNA to LRBP. Based on these results, we conclude that miR-122 plays a regulatory role in LHCGR expression by modulating LRBP levels during FSH-induced follicle growth.

Regulation of Luteinizing Hormone Receptor mRNA Expression in the Ovary: The Role of miR-122

The expression of luteinizing hormone receptor (LHR) in the mammalian ovary is regulated in response to changes in the secretion of follicle-stimulating hormone and luteinizing hormone by the anterior pituitary, at least in part, through posttranscriptional mechanisms. The steady-state levels of LHR mRNA are maintained by controlling its rate of degradation by an RNA-binding protein designated as LHR mRNA-binding protein (LRBP). LRBP forms a complex with LHR mRNA and targets it for degradation in the p bodies. miR-122, an 18 nucleotide noncoding RNA, regulates the expression of LRBP. Thus, the levels of miR-122 determine the cellular levels of LHR mRNA expression. This phenomenon has been examined during the induction of LHR mRNA expression that occurs during follicle maturation in response to rising levels of FSH. In this situation, miR-122 and LRBP levels decrease as LHR mRNA expression undergoes downregulation in response to preovulatory LH surge. miR-122 expression as well as LRBP levels show robust increases. The mechanism of induction of LRBP by miR-122 has also been discussed.

miR-122 Regulates LHR Expression in Rat Granulosa Cells by Targeting Insig1 mRNA

Luteinizing hormone/chorionic gonadotropin receptor (LHR) expression in the ovary is regulated by a messenger RNA (mRNA) binding protein, which specifically binds to the coding region of LHR mRNA. We have shown that miR-122, a short noncoding RNA, mediates LHR mRNA levels by modulating the expression of LHR mRNA-binding protein (LRBP) through the regulation of sterol regulatory element binding protein (SREBP) activation. The present results show that miR-122 regulates LRBP levels by increasing the processing of SREBP through the degradation of Insig1, the anchoring protein of SREBP. We present evidence showing that mRNA and protein levels of Insig1 undergo a time-dependent increase following the treatment of rat granulosa cells with follicle-stimulating hormone (FSH), which leads to a decrease in LRBP levels. Furthermore, overexpression of miR-122 using an adenoviral vector (AdmiR-122) abolished FSH-induced increases in Insig1 mRNA and protein. We further confirmed the role of Insig1 by showing that inhibition of Insig1 using a specific small interfering RNA prior to FSH treatment resulted in the abrogation of LHR upregulation. Silencing of Insig1 also reversed FSH-mediated decreases in SREBP and LRBP activation. These results show that decreased levels of miR-122 increase Insig1 and suppress SREBP processing in response to FSH stimulation of rat granulosa cells.