Our body is continuously exposed to environmental stress, ranging from UV radiation from the sun to parasitic larvae found in raw pork. UV light induces formation of thymidine dimer, creating distortion in the DNA architecture. Upon each breath we inhale, millions of microbes are simultaneously sucked into our airway, posing a threat to our blood-oxygen transport. Even water is classified as a hazardous agent because water consumption decreases blood salt concentration, leading to an imbalance in blood osmolarity. Despite all these hazardous consequences, why hasn’t multicellular life gone extinct by now? It’s because we possess sophisticated physiology to combat these perpetual threats in the blink of an eye. One intriguing homeostatic mechanism that is employed by all multicellular organisms is the regulation of adult stem cell regeneration and repair. Stem cells are phenomenal due to their remarkable ability to either proliferate indefinitely or differentiate into other progenitor cell types. They are prevalent in tissues that undergo dynamic cycles of apoptosis (programmed cell death) and regeneration. For example, within our dermis resides a population of hair follicle stem cells. These hair follicle stem cells are continuously stimulated to differentiate into a mature hair cell, while old hair tissue is swept away. After World War II, many Japanese residents suffered from major hair loss because the nuclear radiation permanently damaged their hair follicle stem cells. Maintenance of tissue architecture through stem cell regeneration and repair is a critical form of homeostasis.
Researchers in Professor Brigitte Gomperts’ lab are interested in the airway basal stem cells (ABSCs) that populate the airway epithelium along the respiratory tract. With the rise of global air pollution due to increased amounts of smoking and radiation, our community is continuously exposed to air contaminants. One critical byproduct of these contaminations is something called reactive oxygen species (ROS)—a mixture of superoxide radicals, hydrogen peroxide, and hydroxyl radicals that causes significant DNA damage and the disruption of cellular metabolism. Gomperts’ Lab has been elucidating the molecular basis of how ABSCs regulate downstream signaling cascades to activate proliferation and repair upon exposure to exogenous ROS.
First, Gomperts’ lab characterized whether ROS concentration flux is necessary to regulate ABSC proliferation and self-renewal. Using fluorescence-activated cell sorting (FACS), mouse ABSCs were sorted out according to their ROS levels. FACS is a molecular technique specialized to segregate a mixture of cell populations into specific subpopulations based on specific characteristics (e.g. surface markers, fluorescent reporter expression, etc). Researchers focused on two subpopulations: ROSLO (ABSCs with low ROS levels) and ROShi (ABSCs with high ROS levels). Both of these subpopulations were then subjected to the in vitro tracheosphere assay, which provides a quantitative readout of cell proliferation and self-renewal based on the size and number of sphere formation. The ROSLO subpopulation yielded higher number and larger tracheospheres compared to the ROShi subpopulation, suggesting that ABSCs with low ROS levels are primed for greater capacity for proliferation and self-renewal than ABSCs with high ROS levels. Thus, Gompert’s lab hypothesized that the ability of ABSCs to self-renew and proliferate is diminished with increasing ROS flux. Researchers added an antioxidant (chemical that eliminates ROS) N-acetyl cysteine (NAC) to both ROSLO and ROShi subpopulations, followed by a rescue of ROS (e.g.H2O2). Lowering ROS in the ROSLO cells diminished their proliferation, but increased proliferation in ROShi cells. But the addition of H2O2 rescued their original phenotypes. Altogether, this suggests that ROS flux from low to moderate levels contributes to ABSC proliferation and self-renewal in vitro.
Now that the Gomperts’ had lab elucidated one major correlation, they pondered whether ROS flux may be linked to the cellular and molecular mechanisms that also regulate ABSC proliferation and self-renewal. They considered some findings from other labs, which showed that a particular protein seems to respond to ROS stress by regulating antioxidant activity of the cell. Precisely, Nuclear factor erythroid-2 related factor (Nrf2) has been shown to regulate expression of antioxidant proteins (e.g. NQO1) in response to ROS stress. They thought that perhaps there might be a link between ROS flux-dependent ABSC proliferation and Nrf2 activity. Nrf2-/- mouse model exhibited higher ROS levels in ABSCs than wildtype control mice. This makes sense, because the presence of Nrf2 acts to reduce ROS stress. Using the same tracheosphere assay, they showed that Nrf2-/- ABSCs manifest reduced proliferation. Recall that ROShi cells also showed less proliferative potential compared to ROSLO cells. This suggests that the NRF1 ROS regulator may be part of the switch that responds to ROS by decreasing the ABSC cells’ ability to proliferate and self-renew.
Furthermore, Gomperts’ lab investigated another crucial signaling pathway regulated by Nrf2. Literature has shown that Nrf2 acts on a downstream pathway called Notch signaling, which is ubiquitous in major developmental pathways. Additionally, it has been shown that the perturbation of Notch signaling results in alterations of ABSC proliferation and self-renewal. Could Notch signaling mediate ABSC proliferation and self-renewal in the context of Nrf2-dependent ROS regulation? Researchers performed Nrf2-knockdown on cell culture and examined the expression of Notch-dependent components involved in ABSC repair upon injury. They observed that the Nrf2-knockdown culture showed less activity of Notch pathway proteins as opposed to wildtype cultures. Altogether, the Gompert’s lab has mapped out a molecular model that, so far, connects ROS flux to ABSC proliferation in the context of Nrf2-dependent Notch activation. To summarize, when ABSCs are exposed to increasing flux of ROS levels, Nrf2 is activated to initiate the expression of antioxidant proteins as well as components of the Notch signaling pathway that regulates ABSC proliferation and self-renewal. As sophisticated as this mechanism may sound, it is only one of thousands of tactics that our cells employ to successfully combat extracellular stress. Our body is much more powerful than we can imagine!
Why do these discoveries bring attention to so many scientists? There is major significance to why people do experiments. Generating these transgenic organisms isn’t always easy, and researchers always encounter failures on a daily basis. But much of the driving force that triggers inquisitive scientists to continue performing research is the search for potential new drug targets. Even though drugs are available in the market, they’re not always perfect. Cost can be a major obstacle to consumers, and the drug itself may produce side effects. Dr. Gomperts has contributed to the scientific community with new knowledge. This now allows researchers in similar fields to explore the signaling systems in greater detail and design new drugs that can be more cost-effective and less risky. If a patient is suffering from lack of ABSC self-renewal due to misregulation between Nrf2 and its targets, perhaps a drug that mimics the structure of these target proteins can be presented to the patient in order to combat exogenous ROS formation. We owe Dr. Gomperts for this remarkable discovery, and we are certainly excited to hear more about her research work in the future!
“Dietary Chemoprevention Strategies for Induction of Phase II Xenobiotic-metabolizing Enzymes in Lung Carcinogenesis: A Review.” Researchgate. Thermo Scientific, n.d. Web.
Hwang, J.; Mehrani, T.; Millar, S. E.; Morasso, M. I. (2008). “Dlx3 is a crucial regulator of hair follicle differentiation and cycling”. Development. 135 (18): 3149–3159. doi:10.1242/dev.022202. PMC 2707782. PMID 18684741
Paul, MK et al. “Dynamic Changes in Intracellular ROS Levels Regulate Airway Basal Stem Cell Homeostasis through Nrf2-Dependent Notch Signaling.” Cell stem cell 15.2 (2014): 199–214. PMC
“Stem Cells in MS.” National Multiple Sclerosis Society. N.p., n.d. Web. 23 Nov. 2016.