Transcriptional upregulation of the p21Cip1 promoter by STAT3 and nuclear ErbB2

Over-expression of the receptor tyrosine kinase ErbB2 is prevalent in approximately 30% of human breast carcinomas and confers Taxol resistance. In breast cancer cells, Taxol induces tubulin polymerization and hyperstable microtubule formation. This in turn prematurely activates Cdc2 kinase allowing early entry into the G2/M phase of the cell cycle resultant in mitotic catastrophe followed by apoptosis. Over-expression of ErbB2 upregulates p21Cip1, which inhibits Cdc2 activation, and leads to Taxol resistance in patients. However, the mechanism of ErbB2-mediated p21 Cip1 upregulation is unclear. Here in this study, we investigated the mechanism of ErbB2 downstream signaling events leading to upregulation. The CDKN1A (p21Cip1) gene promoter contains numerous cis-elements including a Signal transducer and activator of transcription (STAT) Inducable Element (SIE) located at -679 kb. Our studies showed ErbB2 overexpressing cells had increased activated levels of STAT3, and therefore we hypothesized that STAT3 is responsible for the upregulation of the p21Cip1 promoter by ErbB2. EMSA and ChIP assays confirmed the binding of STAT3 to the p21Cip1 promoter and luciferase assays showed higher p21 Cip1 promoter activity in ErbB2 over-expressing transfectants when compared to parental cells, in a STAT3 binding site dependant manner. Additionally, reduced level of STAT3 led to reduced p21Cip1 protein expression and promoter activity indicating that both the STAT3 binding site and STAT3 protein are required for ErbB2-mediated p21Cip1 upregulation. Further investigation of ErbB2 downstream signaling showed increased Src kinase activity in ErbB2 over-expressing cells which was required for ErbB2-mediated STAT3 activation and p21Cip1 increase. Treatment of ErbB2 over-expressing resistant cells with STAT3 inhibitor peptides sensitized the cells to Taxol. In addition to classical signal transduction pathways, I identified a novel ErbB2 mediated regulatory mechanism of p21Cip1. I found that a nuclear ErbB2 and STAT3 complex binds directly to the p21Cip1 promoter offering a non-classical mechanism of p21Cip1 promoter regulation. These data suggest that ErbB2 over-expression can confer Taxol resistance of breast cancer cells by transcriptional upregulation of p21 Cip1 via activation of STAT3 by Src kinase and also by cooperation with nuclear ErbB2. The data suggest a potential clinical mechanism for STAT3 inhibitors in sensitizing ErbB2 over-expressing breast cancers to Taxol. ^

Characterization of glucocorticoid receptor (NR3C1) gene polymorphisms and a family-based association study with hypertension

Hypertension is usually defined as having values of systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg. Hypertension is one of the main adverse effects of glucocorticoid on the cardiovascular system. Glucocorticoids are essential hormones, secreted from adrenal glands in circadian fashion. Glucocorticoid's effect on blood pressure is conveyed by the glucocorticoid receptor (NR3C1), an omnipresent nuclear transcription factor. Although polymorphisms in this gene have long been implicated to be a causal factor for cardiovascular diseases such as hypertension, no study has yet thoroughly interrogated the gene's polymorphisms for their effect on blood pressure levels. Therefore, I have first resequenced ∼30 kb of the gene, encompassing all exons, promoter regions, 5'/3' UTRs as well as at least 1.5 kb of the gene's flanking regions from 114 chromosome 5 monosomic cell lines, comprised of three major American ethnic groups—European American, African American and Mexican American. I observed 115 polymorphisms and 14 common molecularly phased haplotypes. A subset of markers was chosen for genotyping study populations of GENOA (Genetic Epidemiology Network of Atherosclerosis; 1022 non-Hispanic whites, 1228 African Americans and 954 Mexican Americans). Since these study populations include sibships, the family-based association test was performed on 4 blood pressure-related quantitative variables—pulse, systolic blood pressure, diastolic blood pressure and mean arterial pressure. Using these analyses, multiple correlated SNPs are significantly protective against high systolic blood pressure in non-Hispanic whites, which includes rsb198, a SNP formerly associated with beneficial body compositions. Haplotype association analysis also supports this finding and all p-values remained significant after permutation tests. I therefore conclude that multiple correlated SNPs on the gene may confer protection against high blood pressure in non-Hispanic whites. ^

Mechanisms by which nonsense codons downregulate T-cell receptor transcripts

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). Two out of three T-cell receptor β (TCRβ) transcripts carry PTCs as a result of error-prone programmed rearrangements that occur at this locus during lymphocyte maturation. PTCs decrease TCRβ mRNA levels to a much greater extent than mRNAs transcribed from non-rearranging genes. This robust decrease in TCRβ mRNA levels is not a unique characteristic of the T-cell environment or the TCRβ promoter. The simplest explanation for this is that PTC-bearing TCRβ mRNAs elicit a stronger NMD response. An alternative explanation is NMD collaborates with another mechanism to dramatically decrease PTC-bearing TCRβ mRNA levels. ^ In my dissertation, I investigated the molecular mechanism behind the strong decrease in TCRβ mRNA levels triggered by PTCs. To determine the location of this response, I performed mRNA half-life analysis and found that PTCs elicited more rapid TCRβ mRNA decay in the nuclear fraction, not the cytoplasmic fraction. Although decay was restricted to the nuclear fraction, PTC-bearing TCRβ transcript levels were extremely low in the cytoplasm, a phenomenon that I named the nonsense-codon induced partitioning shift (NIPS). I established that NIPS shares several qualities with NMD, including its dependence on translation and NMD factors. Several lines of evidence suggested that NIPS results from PTCs eliciting retention of TCRβ transcripts in the nuclear fraction. This retention, as well as rapid TCRβ mRNA decay, most likely occurs in either the nucleoplasm or the outer nuclear membrane, based on analysis of nuclear and cytoplasmic markers in the highly purified nuclei I used for my studies. To further address the location of decay, I asked whether nuclear or cytoplasmic RNA decay factors mediated the destruction of PTC-bearing mRNAs. My results suggested that a nuclear component of the 3'-to-5' exosome, as well as an endonucleolytic activity, are involved in the destruction of PTC-containing TCRβ mRNAs. Individual endogenous NMD substrates had differential requirements for nuclear and cytoplasmic exonucleases. In summary, my results provide evidence that PTCs trigger multiple mechanisms involving multiple decay factors to remove and regulate mRNAs in mammalian cells. ^

Syntaxin 6- and microtubule- mediated intracellular trafficking contributes to Golgi and nuclear translocation of EGFR

Receptor-mediated endocytosis is well known for its degradation and recycling trafficking. Recent evidence shows that these cell surface receptors translocate from cell surface to different cellular compartments, including the Golgi, mitochondria, endoplasmic reticulum (ER), and the nucleus to regulate physiological and pathological functions. Although some trafficking mechanisms have been resolved, the mechanism of intracellular trafficking from cell surface to the Golgi is not yet completed understood. Here we report a mechanism of Golgi translocation of EGFR in which EGF-induced EGFR travels to the Golgi via microtubule (MT)-dependent movement by interacting with dynein and fuses with the Golgi through syntaxin 6 (Syn6)-mediated membrane fusion. We also demonstrate that the Golgi translocation of EGFR is necessary for its consequent nuclear translocation and transcriptional activity. Interestingly, foreign protein such as bacterial cholera toxin, which is known to activate its pathological function through the Golgi/ER retrograde pathway, also utilizes the MT/Syn6 pathway. Thus, the MT, and syntaxin 6 mediated trafficking pathway from cell surface to the Golgi and ER defines a comprehensive retrograde trafficking route for both cellular and foreign molecules to travel from cell surface to the Golgi and the nucleus.

Stability and potential application of spliced nuclear pre -mRNA introns

A major portion of this thesis work was dedicated to study the nature and significance of spliced introns. The initial work was focused on studying the IVS1$\sb{\rm C\beta 1}$ intron from a T-cell receptor (TCR)-$\beta$ gene. Compared to an intron lariat control from adenovirus pre-mRNA that was spliced in vitro, IVS1$\sb{\rm C\beta 1}$ was debranched less efficiently by HeLa S100 extracts, although IVS1$\sb{\rm C\beta 1}$ also used the consensus branchpoint in vivo. Subcellular-fractionation analysis showed that most IVS1$\sb{\rm C\beta 1}$ lariats cofractionated with pre-mRNA in the nucleus, consistent with the possibility that intron degradation releases splicing factors which will be available for further rounds of splicing. The half-life of IVS1$\sb{\rm C\beta 1}$ from the endogenous TCR-$\beta$ gene was measured using the general transcription inhibitor actinomycin D to be about $\sim$15 min, which was similar to that of unstable mRNAs such as c-myc mRNA.^ The general transcription inhibitor DRB was also used for intron stability analysis. Unexpectedly, DRB decreased intron and pre-mRNA levels only initially, it later increased the levels of intron-containing RNAs. Inhibition of transcription initiation appeared to be the major early effect (the reduction phase); whereas enhanced premature transcription termination was dominant later (the induction phase).^ Having established the procedures for studying in vivo spliced introns, this approach was applied to study the mechanism of nonsense-mediated downregulation (NMD), a phenomena in which premature termination codons (PTCs) decrease the levels of mRNAs. In this study, the novel intron-oriented approach was applied to study the mechanism of NMD. The levels of spliced introns immediately upstream and downstream of a PTC-bearing exon in a TCR-$\beta$ gene were identified and analyzed along with their pre-mRNA. Although PTC reduced the mRNA levels by 4 to 9 fold, the steady-state levels of spliced introns and the pre-mRNA-to-intron ratios were not significantly altered, indicating that the PTC did not significantly inhibit TCR-$\beta$ RNA splicing. Consistent with this conclusion, the half-lives of the PTC$\sp+$ and PTC$\sp-$ pre-mRNA were similar. The protein synthesis inhibitor cyclohexmide (CHX) upregulated the levels of the PTC$\sp+$ mRNA over 10 fold without affecting the levels of the spliced introns, suggesting that the reversal effect of CHX was through stabilization, not production. These results indicated that inhibition of splicing could not be the major mechanism for the NMD pathway of the TCR-$\beta$ gene, instead, suggesting that mRNA destabilization may be more important. (Abstract shortened by UMI.) ^

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^