Molecular dissection of Bruton’s tyrosine kinase signaling in hematopoietic cells using RNAi

RNA mediated gene silencing, RNA interference (RNAi), has recently been identified in mammalians. This thesis describes the utilization and development of RNAi based tools for signal transduction research. The effects of Bruton s tyrosine kinase (Btk) down-regulation with RNAi have been studied with gene expression profiling in hematopoietic cells.

Btk is indispensable for the B cell development. The mutation in the Btk gene causes primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Less is known about the Btk s role in other hematopoietic cells.

This work addresses Btk s role in monocytes, macrophages and mast cells. In paper I, Btk s down-regulation with short interfering RNA (siRNA) caused decreased histamine secretion in the RBL-2H3 mast cell line. Moreover, the concept of using three siRNA against same target gene for enhanced down regulation was introduced for the first time. In paper II, U937 cell line expressing short hairpin RNA (shRNA) against Btk was created. The outcome of stable Btk down-regulation was analyzed with gene expression profiling. Btk was found to regulate 58 transcripts in macrophages (PMA stimulated U937 cells) compared to 11 in monocytes. The analysis suggests Btk s involvement in macrophage effector functions. In paper III, an inducible shRNA vector system was developed to overcome the shortcomings of constitutively expressed shRNAs. The system uses Cre recombinase-mediated site-specific recombination, H1 polymerase III promoter-driven expression and lentiviral delivery. The advantages of pLIND (LentiINDucible) vector system are, no basal shRNA expression in the inactive state, EGFP selection marker and lentiviral transduction enabling gene transfer into primary and other difficult-to-transfect cells. In paper IV, the potential Btk dependency of bone marrow-derived mast cells (BMMC) was addressed. Btk deficient BMMC were shown to grow slower compared to parental cells. This was found to be due to G2/M arrest. Moreover, it was found that Btk negatively regulates expression of novel G protein coupled receptor GPR177. This negative regulation was also found in B cells. Another affected transcript was up-regulation of melanoma antigen (Mela), which is of interest owing to Btk s role as a putative tumor suppressor gene.

List of scientific papers

I. Heinonen JE, Smith CI, Nore BF (2007). "Silencing of Brutons tyrosine kinase (Btk) using short interfering RNA duplexes (siRNA)." FEBS Lett 527(1-3): 274-8
https://pubmed.ncbi.nlm.nih.gov/12220673

II. Heinonen JE, Nore B.F, Lindvall JM, Smith CIE (2007). "Stable silencing of Brutons tyrosine kinase (Btk) in the shRNA expressing human promonocytic cell line U937." (Manuscript)

III. Heinonen JE, Mohamed AJ, Nore BF, Smith CI (2005). "Inducible H1 promoter-driven lentiviral siRNA expression by Stuffer reporter deletion." Oligonucleotides 15(2): 139-44
https://pubmed.ncbi.nlm.nih.gov/15989428

IV. Heinonen J.E, Nyström Lansner K, Blomberg KEM, Lindvall JM, Mattingsdal M, Berglöf A, Grandien A, Smith CIE (2007). "Expression of novel G-protein coupled receptor 177 is negatively regulated by Brutons tyrosine kinase (Btk) in primary bone marrow-derived mast cells." (Manuscript)