Research Article

LMBR1L regulates lymphopoiesis through Wnt/β-catenin signaling

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Science  10 May 2019:
Vol. 364, Issue 6440, eaau0812
DOI: 10.1126/science.aau0812

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Wnt signaling out on a limb

The Wnt/β-catenin signaling pathway is central to hematopoietic stem cell (HSC) survival, renewal, and differentiation. Both excessive and insufficient β-catenin can have adverse consequences, so “destruction complexes” ensure its precise regulation. Using a mouse forward genetic screen, Choi et al. identified a mutation in the gene limb region 1–like (Lmbr1l) that severely impaired lymphocyte development and function. LMBR1L interacted with multiple members of the Wnt/β-catenin signaling pathway. The authors found that LMBR1L works with an endoplasmic reticulum–localized ubiquitin ligase to serve as a second brake on this system in lymphocytes. Deleting β-catenin decreased apoptosis and restored proliferative capacity in an Lmbr1l-deficient mouse T cell line.

Science, this issue p. eaau0812

Structured Abstract

INTRODUCTION

Wnt/β-catenin signaling is a key regulator of mammalian development. In the hematopoietic system, Wnt/β-catenin signaling promotes the survival and renewal of hematopoietic stem cells (HSCs) and the commitment to and differentiation into hematopoietic progenitor and lymphocyte lineages. However, the phenotypic effects of Wnt/β-catenin are not straightforward, in that either excessive or insufficient β-catenin activity has deleterious consequences. For example, combinations of hypomorphic or null adenomatous polyposis coli (Apc) alleles resulting in a gradient of Wnt/β-catenin signaling levels cause either HSC expansion at low levels of activation or depletion of the HSC pool at high levels of activation. Other reports indicate that the activation of the Wnt/β-catenin pathway can promote apoptosis in lymphocytes and decrease quiescence in HSCs.

RATIONALE

In a mouse forward genetic screen for mutations affecting lymphopoiesis and immunity, we identified a recessive hypomorphic mutation in the limb region 1–like gene (Lmbr1l) that resulted in reduced T cell frequencies in the blood. Further phenotypic analysis of Lmbr1l mutant mice demonstrated severely impaired development of all lymphoid lineages, compromised antibody responses to immunization, and reduced cytotoxic T lymphocyte killing activity, natural killer (NK) cell function, and resistance to mouse cytomegalovirus infection. LMBR1L-deficient T cells are predisposed to apoptosis and die in response to antigen-specific or homeostatic expansion signals. We also found impairments in the differentiation of HSCs into the lymphoid-primed multipotent progenitor (LMPP) and common lymphoid progenitor populations that give rise to T cells, B cells, and NK cells. We investigated the molecular function of Lmbr1l in lymphocyte development.

RESULTS

LMBR1L contains nine transmembrane-spanning domains and, in cell fractionation experiments, was most abundant in the endoplasmic reticulum (ER) fraction. LMBR1L coimmunoprecipitated with numerous components of the Wnt/β-catenin signaling apparatus, including ZNRF3, LRP6, β-catenin, glycogen synthase kinase–3α (GSK-3α), and GSK-3β, and with proteins involved in ER-associated degradation (ERAD) of misfolded proteins [UBAC2, transitional ER adenosine triphosphatase (TERA), UBXD8, and glycoprotein 78 (GP78)]. Primary CD8+ T cells from Lmbr1l−/− mice displayed increased levels of β-catenin and the mature forms of Wnt receptor FZD6 and co-receptor LRP6. These effects resulted from the failure of a LMBR1L-GP78-UBAC2 complex to signal the degradation of β-catenin, FZD6, and LRP6 in the ER. Similarly increased expression of β-catenin and mature FZD6 and LRP6 was detected in Gp78−/− primary CD8+ T cells. In addition, reduced protein expression of multiple “destruction complex” components [Axin1, DVL2, β-TrCP, GSK-3α and -3β, and casein kinase 1 (CK1)] was observed in Lmbr1l−/− primary CD8+ T cells. Notably, the knockout of β-catenin in Lmbr1l−/− EL4 cells, a T lymphoma cell line, restored the proliferative potential to a substantial extent and decreased apoptosis caused by LMBR1L deficiency.

CONCLUSION

Absent LMBR1L, Wnt/β-catenin signaling is aberrantly activated, leading to impaired development and functional defects of all lymphoid lineages in mice. Perhaps because of the need for its precise regulation, a prominent element of the Wnt/β-catenin pathway is the canonical destruction complex that ensures the cytoplasmic destruction of β-catenin in the absence of Wnt ligand. Our findings reveal another component of this braking system: an ER-localized LMBR1L-GP78-UBAC2 complex responsible for the degradation of FZD and LRP6 in lymphocytes. Although the upstream signal(s) directing the activation of the LMBR1L-GP78-UBAC2 complex are unknown, we hypothesize that the interface of this complex with the canonical destruction complex synchronizes the activities of these complexes and circumvents leaky inhibition by a single braking mechanism.

LMBR1L plays an essential role in lymphocyte development by limiting Wnt/β-catenin signaling.

A mutation in Lmbr1l caused the impaired development and function of lymphocytes in a forward genetic screen of mice with N-ethyl-N-nitrosourea (ENU)–induced mutations. Development to the LMPP stage and all subsequent lymphoid-directed stages was impaired in LMBR1L-deficient mice. A LMBR1L-GP78-UBAC2 complex in the ER stabilizes the β-catenin destruction complex and promotes the ubiquitin (Ub)–mediated degradation of β-catenin and Wnt receptors. LP, lymphoid progenitor; APC, adenomatous polyposis coli protein; P, phosphoryl.

Abstract

Precise control of Wnt signaling is necessary for immune system development. In this study, we detected severely impaired development of all lymphoid lineages in mice, resulting from an N-ethyl-N-nitrosourea–induced mutation in the limb region 1–like gene (Lmbr1l), which encodes a membrane-spanning protein with no previously described function in immunity. The interaction of LMBR1L with glycoprotein 78 (GP78) and ubiquitin-associated domain–containing protein 2 (UBAC2) attenuated Wnt signaling in lymphocytes by preventing the maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum and by stabilizing “destruction complex” proteins. LMBR1L-deficient T cells exhibited hallmarks of Wnt/β-catenin activation and underwent apoptotic cell death in response to proliferative stimuli. LMBR1L has an essential function during lymphopoiesis and lymphoid activation, acting as a negative regulator of the Wnt/β-catenin pathway.

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