Characterization Of The Er Stress Sensor Ire1α In Mouse And Human Keratinocytes Expressing Ras
Open Access
- Author:
- Craig-lucas, Alayna Brenna
- Area of Honors:
- Toxicology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Adam Bleier Glick, Thesis Supervisor
Gary H. Perdew, Thesis Honors Advisor - Keywords:
- Ras
Keratinocytes
IRE1a
Endoplasmic Reticulum - Abstract:
- The Endoplasmic Reticulum (ER) is the site of protein synthesis for secreted and membrane bound proteins. When a cell accumulates incorrectly folded proteins it leads to a condition called “ER stress”. ER stress is caused by a variety of disease states including cancer and metabolic imbalance. This stress triggers a variety of responses cumulatively called the “Unfolded Protein Response” (UPR). In eukaryotes the three signaling pathways that activate this response are mediated by the ER associated proteins IRE1α, PERK, and ATF6α. For the purpose of this thesis the focus will be the IRE1α pathway primarily because its role in cancer is not well understood. IRE1α is a bi-functional protein with kinase and ribonuclease activities. Upon activation by autophophorylation, the IRE1α nuclease splices a 26 nucleotide sequence from the mRNA of x-Box-binding-protein1 (XBP1), creating the transcript for XBP1s, a transcription factor. The spliced protein helps reduce ER stress by upregulating the expression of genes that encode ER chaperone proteins as well as proteins in the ER-Associated Degradation (ERAD) pathway. This pathway is responsible for delivering misfolded proteins to the proteasome for degradation. The IRE1α nuclease also degrades other ER associated mRNAs in a process called “Regulated IRE1α-Dependent Decay Pathway (RIDD).” This pathway is independent of XBP1s and has been found to increase changes in numerous cell processes including inflammatory responses, apoptosis, growth, metabolism, and macroautophagy. In studies of cellular response to ER stress, the IRE1α nuclease has been found to determine cell fate as it is believed that XBP1s promotes cell survival while the RIDD pathway promotes apoptosis. With low stress, cell survival is mediated by IRE1α nuclease favoring XBP1 splicing while at higher stress levels RIDD activity causes cells to die. Here we have worked to determine if similar opposing effects of IRE1α nuclease regulate cellular response to Ras oncogene activation that occur at the early stages of cancer. The Ras oncogene is an important growth regulatory gene that is mutated in many human cancers. For these studies we have introduced a viral Ras oncogene into primary mouse keratinocytes as well as normal human epidermal keratinocytes to determine the effect on IRE1α pathway activation.