Woodgett, J. R. | Search bioRxiv

bioRxiv2019

Regulation of thymocyte β-selection, development and positive selection by glycogen synthase kinase-3

Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine kinase, that exists as two isoforms in mammals, GSK-3 and GSK-3{beta}, that are key downstream mediators of the phosphatidylinositol 3 kinase, Wnt, Notch and other pathways. Here, we report that simultaneous inactivation of both GSK-3 and GSK-3{beta} during early thymocyte ontogeny has profound effects on both {beta}-selection and positive selection, key checkpoints essential to producing functionally mature {beta} T cells. Conditional GSK-3/{beta} knockout animals (LckCre+ GSK-3{beta}fl/fl) possessed pre-double positive (pre-DP) thymocytes (CD4-CD8-CD117-CD25-) with compromised TCR{beta} chain expression along with elevated levels of {beta}-catenin and reduced Notch activity. {beta}-selection was impaired allowing pre-DP thymocytes to differentiate to DP thymocytes (CD4+CD8+) while bypassing strict requirements for productive TCR{beta} chain rearrangements and functional expression. Also impaired was the requisite pre-TCR and Notch-mediated expansion that normally precedes differentiation to the DP stage. Consequently, LckCre+ GSK-3{beta}fl/fl mice initially generated fewer DP thymocytes that expressed significantly reduced levels of mature TCR. The aberrant DP thymocytes expressed high levels of the pro-survival Bcl-2 family member Mcl-1, failed positive selection and accumulated as CD4hiCD8lo positive selection intermediates resulting in loss of both mature CD4 and CD8 lineages. LckCre+ GSK-3{beta}fl/fl mice succumbed to oligoclonal peripheral lymphomas with high penetrance. These data reveal essential roles for GSK-3 in several checkpoints of early T cell development.

immunology↗

bioRxiv2019

Development of a drug repressible β-catenin mutant mouse to examine effect of activated β-catenin signaling on long-term maintenance of mammary tumor growth

Stabilized {beta}-catenin expression is a well described initiator of mammary tumorigenesis in the mouse and elevated nuclear expression of this protein has been observed in human triple-negative tumor samples. However, the importance of stabilized {beta}-catenin to continued tumor growth after initiation, and in the context of other driver mutations, has yet to be elucidated. To ascertain the importance of stabilized {beta}-catenin after tumor initiation, we generated a novel transgenic mouse model, utilizing the tet-off system, to control expression of a stabilized mutant of {beta}-catenin. Pups from early litters carrying one allele of regulatable stabilized {beta}-catenin at the Rosa26 locus, but not targeted by Cre, were smaller in size compared with wildtype littermates and also developed skin lesions, requiring euthanasia. Maintenance of breeding cages on doxycycline chow allowed for healthy transgenic pups to survive past 3 weeks of age and 6 animal cohorts were established. We used two different mammary Cre strains, WAP-Cre and MMTV-NLST-Cre, crossed with regulatable, stabilized {beta}-catenin (bcatFL) animals, as well as animals expressing mutated p53-R270H. While mammary tumors developed in animals expressing Cre-targeted regulatable bcatFL, the penetrance and growth kinetics were lower than observed with other mammary mouse models expressing stabilized {beta}-catenin. Since fewer animals developed tumors, we focused analysis on the effect of turning off stabilized {beta}-catenin expression following tumor initiation. In the animals examined, a delay in tumor growth, but not regression, was observed in animals expressing Cretargeted bcatFL as well as those expressing both bcatFL and p53-R270H. These results indicate that in a less aggressive {beta}-catenin model, additional mutations likely provide independence from the initiating event.

cancer biology↗