The proprotein convertase subtilisin/kexin type 9 (PCSK9) was first characterized in 2003 by Seidah and colleagues and marked the beginning of what is now considered by many as the greatest advancement in the field of cardiovascular disease (CVD) research since the discovery of the LDLR nearly half of a century ago. Since its discovery, PCSK9 was shown to enhance the degradation of cell-surface low-density lipoprotein (LDL) receptor (LDLR) and gain-of-function (GOF) mutations were shown to correlate with CVD risk. In contrast, patients carrying loss-of-function (LOF) mutations in PCSK9 highlighted a novel therapeutic approach for LDL lowering as they exhibit a life-long state of hypocholesterolemia and reduced CVD risk. A decade after the cloning of the PCSK9 gene, pharmaceutical companies have now developed a variety of PCSK9 inhibitors, ranging from monoclonal antibodies (mAbs) to small interfering RNA (siRNA) and vaccines, which have been shown to markedly reduce LDL cholesterol levels in pre-clinical models, as well as in patients at high risk of CVD. Despite these advances, there remained several unanswered questions regarding the mechanisms by which PCSK9 expression and secretion is regulated in the liver; the tissue from which the circulating pool of PCSK9 almost exclusively originates. The thought that further development of our understanding of PCSK9 biology may lead to the discovery of a signaling cascade that could be targeted by small molecules, the only class of inhibitor that has not yet been developed, has now merited additional research attention.
The focal point of my doctoral studies represents the axis between a cellular process known as endoplasmic reticulum (ER) stress and PCSK9 expression/biosynthesis. ER stress is a deleterious cellular process that is known to occur in secretory cell types, such as liver hepatocytes, and is a well-established causative driver of an array of human diseases ranging from CVD to neurodegenerative diseases. ER stress is prevalent in the livers of patients with metabolic disease and is also known to activate the transcription factor capable of regulating PCSK9 levels, the sterol regulatory element-binding protein 2 (SREBP2). Based on this information, the first aim during the course of my PhD studies was to determine whether ER stress affected the expression and secretory status of PCSK9. In the past several years, I demonstrated that ER stress caused by ER Ca2+ depletion led to a marked increase in PCSK9 protein expression, but blocked its secretion as a result of its retention in the ER. Such a result was also associated with heightened hepatic LDLR expression and reduced LDL cholesterol levels in mice. Additional studies also characterized a variety of agents, including caffeine, as potent inhibitors of PCSK9 expression via increasing ER Ca2+ levels, which antagonized SREBP2 activity. As our initial studies revealed ER PCSK9 retention as a viable strategy for PCSK9 inhibition and LDL lowering, follow-up studies were also carried out to determine the outcome of such a strategy on liver function and injury. Given that heritable mutations in proteins that transit the ER can accumulate in this compartment and cause ER storage disease (ERSD), it was critical to further evaluate whether ER PCSK9 retention would lead to a similar outcome.
In a series of experiments with rather surprising outcomes, we observed that the retention of the LOF Q152H PCSK9 mutant in the ER failed to cause ER stress; even in mice overexpressing the protein. Interestingly, tissue culture and mouse models demonstrated that the retention of PCSK9 in this cellular compartment increased the cellular abundance of ER stress response chaperones, such as the glucose-regulated proteins of 78- and 94-kDa (GRP78 and GRP94, respectively), but did not activate transducers of the ER stress signaling cascade. Strikingly, mice expressing the ER-retained PCSK9 Q152H mutant were protected against ER stress, suggesting a novel co-chaperone-like role of intracellular PCSK9. Collectively, the ER environment including secondary messengers like Ca2+ as well as its chaperones, plays a critical regulatory role on PCSK9 expression and secretion. Agents that increase ER Ca2+ levels can be utilized to block PCSK9 expression at the mRNA level to increase hepatic LDL clearance, and ER PCSK9 retention may also represent a safe avenue with a similar LDL lowering outcome. Beyond LDL lowering, hepatic ER PCSK9 retention may also serve as a novel strategy to enhance ER function and protect against ER stress-driven diseases of the liver. / Thesis / Doctor of Philosophy (Medical Science)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/25329 |
| Date | January 2019 |
| Creators | Lebeau, Paul |
| Contributors | Austin, Richard, Medicine |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0024 seconds