Probing the Role of Insulin-Like Growth Factor-Binding Protein 3 and Humanin in Regulating Hyaluronan Function - PROJECT SUMMARY Glycosaminoglycans (GAGs), components of the highly dynamic extracellular matrix (ECM), play a crucial role in the undertaking of numerous cellular processes and a variety of human diseases result from disruption of their function. Hyaluronan (HA) is a critical component of the ECM and despite its simple primary structure, it regulates cellular responses in a highly complex manner balanced by contributions from factors that include specific signaling pathways and interactions with cell receptors or other extracellular HA binding proteins. How HA acts as a versatile macromolecule that operates via different mechanisms to regulate distinct downstream signaling and how dysregulation of HA interactions affects cell viability is largely unknown, needs to be elucidated, and currently represents a significant gap in our knowledge. Our long-term goal is to unravel the basic mechanisms by which GAGs function as extracellular molecular switches to regulate cell survival. The overall objective of this proposal is to shed light on novel mechanisms employed by HA in regulating cell signaling. Our central hypothesis is that HA is a key biomolecule that regulates extracellular phosphorylation, neurotransmitter and growth factor receptor signaling, and metabolic reprograming, and that modulation of these mechanisms ultimately affects cell function. Our rationale is that gaining sufficient knowledge of these mechanisms will offer new therapeutic opportunities. Our specific aims are to test the following hypotheses: 1) Binding of HA to IGFBP-3 blocks the protein’s ability to bind humanin which can now bind amyloid beta (Aβ) blocking its phosphorylation on Ser8 by extracellular PKA; 2) Nicotine acting via either the α7-nicotinic acetylcholine receptor or the β-adrenergic receptors activates epidermal growth factor receptor and insulin-like growth factor type 1 receptor to increase PKA activity, increasing HA levels and activation of HA-CD44 signaling, inhibiting p53, increasing the levels of heparanase in the media leading to activation of ecto-casein kinase 2, inhibiting caspase-3 and blocking apoptosis; 3) Hypoxia leads to increased HA levels activating HIF-1α and glycolysis, increasing lactate production which will then activate MMP2 decreasing Aβ40/42 toxicity (A) and MMP9 increasing sE-cad levels (B) and the BDNF/pro-BDNF ratio (C), blocking apoptosis, increasing cell survival and hypoxia-induced cisplatin resistance. This proposal is significant because better understanding of these basic mechanisms will advance our knowledge of diseases resulting from dysregulation of protein-peptide- carbohydrate signaling and innovative because it will investigate heretofore-unexamined HA functions. Expected outcomes will include unraveling HA signaling networks to develop novel therapeutics targeting specific HA- mechanisms. This R15 application will have a positive impact because it will lay the groundwork to develop better approaches to target GAG-related diseases, provide an effective vehicle for introducing undergraduate and graduate students to an authentic and extensive hands-on research training at an early stage of their education, and cultivate student confidence, resilience, appreciation, and interest in biomedical research careers.
