Integrins contribute to lymphopoiesis, whereas Toll-like receptors (TLRs) facilitate the myeloid

Integrins contribute to lymphopoiesis, whereas Toll-like receptors (TLRs) facilitate the myeloid replenishment during inflammation. were cell-intrinsic and could be recapitulated on bone marrow stromal cell culture. Furthermore, defective lymphopoiesis correlated strongly with failure of hematopoietic progenitors to form close contact with stromal cell niche and was not the result of the defect in the assembly of antigen receptor or interleukin-7 signaling. These findings define gp96 as the only known molecular chaperone to specifically regulate T- and B-cell development. Introduction Integrins are a family of 24 heterodimers in vertebrates formed noncovalently by 18 and 8 Rabbit polyclonal to SERPINB9 integrins, of which 17 integrins are expressed in the hematopoietic system.1,2 Known best for their adhesion properties, integrins also orchestrate signals between extracellular matrix and intracellular cytoskeletons in regulating diverse functions of cells, including proliferation and differentiation. However, despite the expression of integrins on hematopoietic stem cells (HSCs) and the role of integrins in HSC homing to the bone marrow (BM) niche, their function in hematopoiesis remains controversial. For example, although 4 integrin has been implicated in both T and B lymphopoiesis from fetal HSCs,3,4 it appears to play a less significant role in adult hematopoiesis.5,6 Furthermore, combined deletion of both 1 and 7 integrins, which are the only known partners of 4 integrin, causes no defect in either lymphopoiesis or myelopoiesis.7 Genetic 2 integrin deficiency causes myeloid hyperplasia, including profound granulocytosis and splenomegaly, but no significant problems in hematopoiesis.8 Clearly, both 4 and 2 integrins are involved in homing of HSCs in the BM and recruitment of leukocytes to sites of inflammation.5,9,10 Although Bay 65-1942 pan-integrin deficient system is now available,11 no resolution of the roles of integrin in hematopoiesis has emerged. Toll-like receptors (TLRs) are pattern recognition receptors that play important roles in sensing pathogen-associated molecular patterns from microbes, which are critical Bay 65-1942 for host immune response.12 More than 10 TLRs have been described in vertebrates, recognizing a spectrum of microbial moieties, such as endotoxin, flagellin, dsRNA, and DNA. In the steady state, TLRs do not contribute significantly to hematopoiesis, although TLRs on HSCs have been implicated in the replenishment/recruitment of myeloid cells in response to inflammation.13,14 TLRs and integrins do not share significant structural homology. Nevertheless, the folding and proper expression of many TLR and integrin family members are dependent on gp96, the heat shock protein 90 (HSP90) paralogue in the endoplasmic reticulum (ER). Deletion of gp96 leads to posttranslational loss of multiple TLRs (TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, and TLR9) and several integrins Bay 65-1942 (2, 4, and V integrins),15C17 although no study has probed the entire hematopoietic system-specific integrins for their dependence on gp96. As a major ER luminal protein whose expression can be further induced by accumulation of misfolded proteins, gp96 is also thought to participate in the ER-unfolded protein response (UPR)18 and ER-associated protein degradation,19 and has been implicated to play a major housekeeping function to maintain protein homeostasis in the secretory pathway.20 The discovery that gp96 seems to selectively fold TLRs and integrins15C17 was unexpected, which raises the intriguing possibility that gp96 is evolved to play more specialized function in the multicellular organism. In this study, we used tamoxifen (TAM)Cinducible gp96 knockout (floxed mice were crossed to R26R-creERT2 mice (kindly provided by James Y. H. Li, University of Connecticut Health Center [UCHC]) and further backcrossed to C57BL/6 background for 6 to 10 generations. Control mice were mice. All mice were maintained by the Center for Laboratory Animal Care of UCHC (Farmington, CT) on an Institutional Animal Care and Use CommitteeCapproved animal care protocol. Cell lines and gp96 mutant 70Z/3 pre-B cells were a gift from Brian Seed (Harvard University),15 which were cultured in RPMI medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (Atlas Biologicals), 55M 2-mercaptoethanol (Invitrogen), and penicillin-streptomycin (Invitrogen). OP9 and OP9-DL1 cells were cultured in -minimum essential medium containing l-glutamine and ribonucleotides (Invitrogen) supplemented with 20% fetal calf serum, 1mM.

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