Theses Doctoral

3-Phosphoinositide-dependent kinase-1 (PDK1)-mediated signaling regulates hematopoietic stem cell (HSC) quiesence by governing the oxidative response

Matsushima, Danielle Erina

Regulation of hematopoiesis through the finite control of hematopoietic stem cell (HSC) quiescence and proliferation is critical to the health of the organism since disturbances in blood production can lead to clinical malignancies such as anemia or leukemia. Therefore, elucidating the processes that govern HSCs is vital to advance our understanding of hematological diseases. Interestingly, HSCs can be regulated through a variety of ways. Extrinsic cues from the niche provide signals that govern HSC quiescence, proliferation, self-renewal, and differentiation. These external signals are converted to internal messages through the use of signal transduction pathways that relay information from the cytoplasm to the nucleus. While many pathways contribute to HSC regulation, the PI3K/AKT/mTOR-pathway is especially pertinent because it has been implicated in cell survival, metabolism, proliferation, and death. Many groups have identified key players within PI3K/AKT/mTOR-signaling that regulate HSC quiescence; however, these studies are hindered by the redundancy of multiple isoforms and compensatory signaling mechanisms by other members within the pathway. PDK1 is considered to be a master regulator of PI3K-signaling because it is directly activated by PI3Ks and can govern activity of a variety of substrates within the PI3K-pathway. Because of this, it is an excellent candidate to fully delineate how PDK1-mediated PI3K-signaling functions to maintain HSC quiescence.
In the current study, conditional deletion of PDK1 in HSCs was achieved through the use of a hematopoietic specific Vav-Cre line. The loss of PDK1 in HSCs led to reduced numbers and an inability to provide radioprotection in primary BMTs. Furthermore, PDK1-deficient HSCs exhibit impaired quiescence and increased cycling. Strikingly, PDK1-mutant HSCs have markedly high levels of reactive oxygen species (ROS), which led to increased proliferation, DNA damage, and apoptosis in progenitor cells. Administration of a ROS scavenger, N-acetyl cysteine (NAC) partially rescued the increased proliferation and differentiation of phenotype Pdk1Hem-KO cells both in vitro and in vivo, suggesting that abnormal HSC activity in PDK1-deficient cells was in part due to increased ROS. Furthermore, mechanistic studies identified a remarkable decrease in the levels of nuclear HIF1α in HSCs. Immunoblots and phosflow studies demonstrated reduced activation of p-p70S6K, a well defined positive regulator, and increased GSK3β, a key negative regulator of the HIF1α protein. These data suggested that ROS levels are unrestrained since HIF1α is not present in Pdk1Hem-KO HSCs to activate transcription of genes that moderate oxidative stress. In addition, Pdk1Hem-KO HSCs also show increased levels of Hif3α and IPAS mRNA, which are believed to inhibit the transcriptional activation of HIF1α. In essence, the results from these experiments are the first to implicate PDK1 as a critical regulator of HSC quiescence and as a moderator of PI3K-signaling to alleviate oxidative stress. In addition, this study is the first to suggest that HIF3α and IPAS may play a role in HSCs.


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More About This Work

Academic Units
Genetics and Development
Thesis Advisors
Rathinam, Chozha Vendan
Ph.D., Columbia University
Published Here
August 1, 2016