Application of TAT-TVTSP as a novel anti-asthmatic drug and molecular mechanism study for Bcl-2 leading to neurite outgrowth

Abstract

Asthma is a chronic lung disease that causes airflow obstruction due to airway inflammation. However, its therapeutics still remain inadequate. We previously reported that phospholipase D1 (PLD1) is required for house dust mite allergen (HDM)-induced airway inflammation. We also revealed that PLD1 is specifically inactivated by binding to TVTSP motif of assembly protein, 180 kDa (AP180). Therefore, we aim to develop a novel anti-asthmatic agent that could suppress airway inflammation by inhibiting PLD1, and examine its acute and chronic toxicity. We designed TAT-TVTSP, a PLD1-inhibitory peptide fused with cell-penetrating peptide (CPP). TAT-TVTSP was efficiently delivered into bronchial epithelial cells, and reduced Dermatophagoides farinae 2 (Der f 2)-induced PLD activation and IL-13 production. We also demonstrated that the TVTSP motif of AP180 directly bound to the His858/Lys860 residues of PLD1. Intranasally-administered TAT-TVTSP was efficiently transferred to murine airway tissues, and ameliorated airway inflammation in HDM-induced allergic asthma mouse model. Moreover, we investigated the safety of TAT-TVTSP as a therapeutic agent through single- and repeated-dose toxicity studies in a mouse model. Taken together, these results indicated that a TAT-TVTSP may be useful for treating HDM-induced allergic inflammatory asthma. Bcl-2 is highly expressed during neural development in the nervous system, and plays a key role in modulating cell survival. Besides the anti-apoptotic function, Bcl-2 was previously suggested as a mediator of neuronal differentiation. However, the mechanisms by which Bcl-2 might have impacts on the neurogenesis is not sufficiently understood. We aim to unveil the non-apoptotic functions of Bcl-2 during neuronal differentiation. We analyzed whole gene expression through microarray from rat neural stem cells, and found that Bcl-2 overexpression induced the expression of various neurogenic genes. Moreover, Bcl-2 overexpression increased neurite length as well as expression of Bmp4, Tbx3, and proneural bHLH transcription factors in H19-7 cells. Thereafter, we demonstrated that Bcl-2 overexpression promoted neurite outgrowth through Bmp4/Tbx3/NeuroD1 cascade in H19-7 cells, indicating that Bcl-2 may have a direct role in neuronal differentiation besides well-known anti-apoptotic function.