Latexin Regulates Stem Cell Numbers Via a NF-eB-Dependent Pathway. (2008)

Abstract Latexin was first discovered in the developing lateral cortex of the brain and is the only known endogenous carboxypeptidase A (CPA) inhibitor in mammals. Our recent studies have revealed that latexin exerted a novel function in regulating murine hematopoietic stem cell number by repressing stem cell self-renewal and enhancing apoptosis (Liang et al., Nat. Gen.39:178, 2007) . However, the underlying mechanism remains obscure. To test whether latexin acts through its canonical mechanism in hematopoietic cells we used fluorescence immunohistochemistry to reveal that latex and CPA did not co-localize intracellularly. Moreover, in contrast with latexin, an unrelated inhibitor of CPA did not affect hematopoietic stem cell numbers in vivo. Thus, latexin appeared to act through a novel pathway unrelated to the inhibition of CPA. To identify novel intracellular protein partners of latexin, tandem affinity purification (TAP) tag-labeled latexin was overexpressed in FDCP1, a primitive hematopoietic progenitor cell line. Latexin-interacting proteins were isolated from lysates of FDCP1-TAP-latexin and FDCP-1-TAP control by TAP. Purified eluates were screened by mass spectrometry proteomic technologies. Eleven proteins, but not CPA, were identified as latexin-interacting partners by LC-MS analysis. Using Western blots and co-immunoprecipitation we found that latexin interacted with a novel binding partner, ribosome protein S3 (RPS3), a moiety recently found to complex with NF-kB and thus regulate its selective activation of target genes (Wan et al. Cell131:927, 2007). We hypothesized that latexin may compete with NF-kB for binding RPS3 and thus indirectly affect NF-kB-directed gene expression. Thus, in order to determine if NF-kB-responsive genes were differentially regulated in primitive hematopoietic cells in response to latexin modulation, we made use of a mouse model congenic for the latexin locus. We introgressed the high-expressing C57BL/6 latexin allele onto the normally low-expressing DBA/2 genetic background. We then compared the gene expression profiles of Lineage negative, Sca-1 positive, and c-kit positive (LSK) cells isolated from the bone marrow of congenic and DBA/2 background (control) mice using microarrays. A functional cluster of genes known to be regulated proximally by NF-kB and which are related to mechanisms of latexin action--namely apoptosis and cell replication—were differentially expressed in concordance with the latexin allele present. These results are consistent with a model in which latexin impeded RPS3-NF-kB complex formation and subsequently altered NF-kB-mediated gene transcription. Taken together, our findings provide a potential mechanism by which latexin regulates hematopoietic stem cell number via a heretofore unappreciated pathway. Liang Y, M Jensen, B Aronow, H Geiger, G Van Zant. The quantitative trait gene latexin influences the size of the hematopoietic stem cell population in mice. Nature Genetics 39:178–88, 2007. Wan F, DE Anderson, RA Bamitz, A Snow, N Bidere, L Zheng, V Hegde, LT Lam, LM Staudt, D Levens, WA Deutsch, MJ Lenardo. Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation. Cell 131:927–39, 2007.