A long-standing goal of the Eckhert Lab has been to understand the biology of boron and its role in human health at the molecular level. The argument for boron's essentiality in humans is weakened by the fact that no molecular mechanism has been identified. Boron is an essential nutrient for plants, and the human consumption of boron from dietary plants equals an intake of approximately 1 mg/day. Boron from plants is nearly completely absorbed as boric acid (BA) and circulates unchanged with a half-life of about 23 hours. This work focuses on three unsolved problems in the area of human boron biology: 1) characterization of the molecular target of BA and how it interacts with protein targets, 2) the signal transduction consequences of BA induced Ca2+ signaling dysregulation, and 3) a molecular explanation of how BA can inhibit cell proliferation without resulting in apoptosis. Collectively, the work presented herein defines a mechanism for BA in human cells.
The Eckhert Lab has previously reported that the molecular target of BA is cyclic ADP-ribose (cADPR), the endogenous agonist of the ryanodine receptor (RyR), an endoplasmic reticulum (ER) Ca2+ channel. There were two questions remaining. First, it was unknown if cADPR binds to RyR or one of its accessory proteins. Second, it was unclear how BA interacted with the RyR complex to inhibit cADPR induced Ca2+ release from the ER. In the first chapter, I provide evidence that demonstrates cADPR binds directly to the accessory protein FKPB12. In this chapter, I also provide evidence that demonstrates that in the presence of BA, the binding of cADPR to FKBP12 is inhibited.
The Eckhert Lab has also reported that BA induces mild ER stress, a consequence of ER Ca2+ dysregulation. Building on the work of Kimberly Henderson and Sarah Kobylewski, I describe in chapter 2 the molecular pathways that are activated by BA treatment of prostate cancer cells at physiological concentrations. I describe evidence that BA activates the eIF2alpha/ATF4 and ATF6 pathways. These results raise two additional questions. EIF2alpha; phosphorylation can occur through one of four kinases, each sensitive to different environmental stresses, but it was not known which kinase is activated by BA. In addition, model activators of the eIF2alpha; pathway usually activate CHOP, an apoptotic protein. However, BA does not cause apoptosis or increase levels of CHOP protein or mRNA and we did not have a molecular explanation for this observation.
The question of which kinase was responsible for BA induction of ph- eIF2alpha; needed to be addressed. In the third chapter, I identify PERK as the kinase sensitive to BA. PERK has two known substrates, eIF2alpha; and Nrf2. I also provide evidence that demonstrates that BA treatment activates Nrf2, which is a transcription factor for the antioxidant response element and induces transcription of several antioxidant genes. This data indicates that BA treatment can increase levels of antioxidant proteins in the cell and provides evidence for why BA does not induce CHOP .
The work described in these three chapters make a large contribution to the field of boron biology. Future studies should explore the link between the molecular mechanisms outlined herein with the specific effects observed with boron supplementation in animal models.