Despite recent breakthroughs in identifying prospective stem cell markers (e.g., LGR5) in the intestines and the gastric antrum, relatively little is known about the stem cells of the gastric (body) corpus epithelium. Our overall goal is t better characterize the corpus stem cell under normal conditions and in response to injury. Preliminary data show that the selective estrogen receptor modulator tamoxifen causes ablation of nearly all parietal cells, the acid-secreting lineage, in the mouse stomach within 3 days. Within 6 hours, parietal cells begin to die (atrophy), and progenitor cells in the isthmal stem cel niche of corpus gastric units begin to expand in response. We further find that: 1) the cell surface protein CD44 is required for normal stem cell homeostasis, because proliferation is halved in Cd44-/- relative to wildtype mice;and 2) CD44 marks the expanding isthmal progenitor population. CD44 activates STAT3 and is induced by ERK signaling. We show here that ERK and STAT3 are activated in early expanding progenitors, and levels of the ERK-induced transcription factor EGR1 peak at 3 days, in concert with maximal progenitor expansion. We hypothesize that parietal cell death leads to signals that activate the ERK pathway in isthmal stem/progenitor cells, which causes increased CD44 expression and increased proliferation.
In Aim 1, we will determine whether ERK signaling is required for normal stem cell homeostasis and atrophy-induced expansion using both ERK pharmacological inhibition and pedigrees of mice with deficient (Egr1-/-) and overactive (Nab2-/-) ERK.
In Aim 2, we will determine whether CD44 is required or sufficient for atrophy-induced progenitor expansion. We will examine kinetics of stem cell expansion in Cd44-/- mice, and we will either activate CD44 in wildtype mice ?tamoxifen by injection of the CD44 ligand Hyaluronan or block CD44- Hyaluronan interactions with the inhibitor PEP-1. We will determine if CD44 induces STAT3 and/or if it is required for progenitor expansion.
In Aim 3, we will isolate vehicle- and tamoxifen-treated CD44-expressing epithelial progenitor cells by FACS and laser-capture microdissection and then use RNA-Seq to generate gene expression profiles of the normal and atrophy-expanded progenitor cells that will then be integrated bioinformatically with our existing database of global gene expression in stem and progenitor cells. We will also perform limiting dilution assays to determine the stem cell frequency within the epithelial CD44 population. In humans, chronic inflammation can cause parietal cell atrophy, which in turn changes stem cell proliferation and differentiation. Atrophy predispose patients to gastric cancer, but changes in stem cells secondary to inflammation/injury are also an important and understudied aspect of adult diseases in multiple other tissues (e.g., hematopoietic stem cell differentiation changes in arthritis). Thus, the experiments proposed may help us begin to understand stem cell response to injury in multiple tissues and diseases. Finally, our new finding that the key drug tamoxifen causes gastric toxicity warrants further study in humans.
The current application proposes several experiments to learn how stem cells in the body (corpus) of the lining of the stomach respond to loss of the acid-secreting parietal cells. Little is known about corpus stem cells, yet the stomach is the site of enormous human disease from incredibly commonly occurring gastric ulcers to gastric cancer, which is the second-leading cause of cancer deaths worldwide. In the United States, gastric cancer affects disadvantaged minorities at around double the rate, and it is particularly prevalent in developing nations. Ultimately, adult stomach diseases, like cancer, occur because injury and/or inflammation cause alterations in how the stem cell behaves. Thus, we propose to kill parietal cells and use molecular, genetic, and gene sequencing techniques to determine how the stem cells respond to this injury, which is what happens in humans in the first stages of progression to cancer. Most of what we know about stem cells - even in tissues like the blood where stem cells are much better understood - concerns how they behave under normal conditions, yet stem cells may behave differently as people age and during chronic inflammatory state, so our experiments may help us extrapolate from the stomach to understand stem cells in tissues other than the stomach. Finally, we will kill parietal cells in these experiments by injecting the estrogen-like molecule tamoxifen, which we show here causes parietal cell death. Tamoxifen prescriptions cost the public around half a billion dollars a year in the US, yet there have been no previous studies of its effects on important organs in animals, so our experiments might help determine whether tamoxifen might carry long term risk for stomach injury in patients.
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