University of Minnesota Ph.D. dissertation. April-2015. Major: Veterinary Medicine. Advisor: Mathur Kannan. 1 computer file (PDF); x, 200 pages.CD38 is a multifunctional enzyme that regulates intracellular calcium ([Ca++]i ) homeostasis. It is expressed in airway smooth muscle (ASM) cells where it elevates [Ca++]i through its enzymatic product cyclic ADPribose (cADPR) and increases ASM contractility. Increased expression of CD38 in the ASM cells derived from the asthmatic patients (AS-HASM) and attenuated airway hyperresponsiveness to contractile stimuli shown by CD38-/- mice implicate the importance of CD38 in asthma. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is considered to be an important mediator for airway pathology in asthma. The reason for the differential expression of TNF-alpha-induced-CD38 in AS-HASM cells, does not involve transcriptional regulation of CD38 which is through signaling pathways mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K), and transcription factors nuclear factor kappa-B (NF-kB) and AP-I. Thus I hypothesized that post-transcriptional regulation of CD38 by microRNAs account for the differential expression of TNF-alpha induced -CD38 in AS-HASM cells. Among the several potential microRNAs predicted for CD38 by microRNA target-predicting algorithms, I selected miR-140-3p and miR-708 for further studies, as these showed differential expression in the AS-HASM cells compared to those from healthy subjects. Overexpression of these either microRNAs in ASM cells inhibited the TNF-alpha induced expression of CD38 at messenger RNA (mRNA) and protein levels. Luciferase-reporter assays with a mutated 3'UTR of the CD38 transcript confirmed the specific target sites for both microRNAs. Transcript stability assays revealed that mRNA degradation is not the mechanism underlying regulation by microRNAs. Examination of the expression and activation levels of proteins in the upstream signaling pathways of CD38 revealed that miR-140-3p, by inactivating p38 MAPK and NF-kB, and miR-708, by inactivating c-Jun N-terminal kinase (JNK) MAPK and Akt by elevating the expression of their phosphatases MKP-1 and PTEN respectively, control the expression of CD38 indirectly. Further, we found that miR-708 downregulates the expression of many chemokines and inhibits the serum induced proliferation of human ASM cells. We conclude that both microRNAs have therapeutic potential in controlling asthma related symptoms through regulating the expression of CD38 and chemokines and controlling the proliferation of human ASM cells
