We describe new roles for the RPTP CD148 in the regulation of SFK activity in the immune system. Our studies of CD148 TM-KO mice showed a phenotype similar to mice deficient in CD45 suggesting that CD148 is a positive regulator of ITAM-dependent signaling in the B cell and myeloid lineages. Importantly, analyses of the signaling pathways in B cells and macrophages from CD45/CD148 DKO mice revealed substantial defects not seen in the singly deficient mice. At the molecular level, DKO B cells displayed impaired calcium flux and MAP kinase phosphorylation. Importantly, CD148 and CD45 are both required to optimally dephosphorylate the C-terminal negative regulatory tyrosine of SFKs, such as Lyn, in B cells. Very similar defects were also observed in Fc receptor-mediated activation and phagocytosis in BMDMs. These data suggest a high level of redundancy between the two structurally distinct RPTPs CD148 and CD45 in regulating SFKs. Moreover, these findings suggest a reinterpretation is needed of the function of CD45 in non-T cell lineages where CD148 is constitutively expressed and may compensate at least partially for some of the described phenotypes seen in the absence of CD45.
One of the striking features of the DKO mice is their early mortality. We speculate the development of the myeloproliferative syndrome with liver and lung infiltration likely contributed to their premature death. Interestingly, the myeloproliferative syndrome is transplantable with BM and can be observed even in aged chimeric mice (data not shown). The animals also develop anemia, which could contribute to their death. However, the precise cause of death could be complex since multiple lineages and pathways may be dysregulated. Further analysis will be aided by the lineage specific inactivation of CD148.
It is tempting to speculate that the observed defects in SFK signaling are responsible for the phenotype. Indeed, many features of the DKO mice resemble those of mice deficient in different combinations of SFKs. A milder myeloproliferative syndrome was also observed in Lyn deficient mice (Harder et al., 2001
), suggesting that the reduced activity of SFKs in CD45/CD148 DKO mice could explain this phenotype. The myeloproliferative disease in DKO mice was accompanied by a severe block in early B cell development in the C57BL/6 genetic background. However, a different phenotype was observed in the Lyn singly-deficient mice, which instead display B-cell hyperactivity and B-cell mediated autoimmunity leading to severe glomerulonephritis (Hibbs et al., 1995
; Nishizumi et al., 1995
). CD45/CD148 deficiency likely affects many SFKs and so the balance between the activities of the individual SFKs governs the outcome. Deficiencies in different combinations of SFKs can result in variable phenotypes, ranging from immunodeficiency (Saijo et al., 2003
) to autoimmunity (Hibbs et al., 1995
; Nishizumi et al., 1995
) and leukemogenesis (Marth et al., 1988
; Marth et al., 1986
). Due to the very limited number of B cells we obtained from DKO mice, we could not address the phosphorylation status of the individual SFKs. However, it is possible that different SFK members are affected to a varying degree by deficiency in each RPTP. In addition to SFKs, there may be other substrates of CD148 and CD45 that contribute to the phenotype we observed in DKO mice.
Our studies of BMDM support a role for CD45 and CD148 in ITAM-receptor mediated signaling in the myeloid lineage. The defective Fc receptor-mediated phagocytosis and TNFα production in macrophages derived from the CD45/CD148 DKO is reminiscent of SFK and Syk deficiency in macrophages. Previous studies of lyn/hck/fgr
triple KO mice demonstrated a defect in FcR-mediated phagocytosis (Fitzer-Attas et al., 2000
). An even more profound defect in FcR-mediated phagocytosis was observed in Syk deficient mice (Crowley et al., 1997
). Thus, it seems likely that the functions of CD45 and CD148 overlap and converge on SFKs in ITAM-dependent BCR- and FcR-mediated functions in B cells and macrophages.
It is intriguing that while DKO mice have a severe myeloproliferative disease they also develope a B cell developmental block and impaired BCR and FcR signaling. The myeloproliferative disease in DKO may involve cytokine- and growth factor receptor-induced pathways, in which CD45 and CD148 may play different roles. While it is possible that these RPTPs directly act on cytokine receptor associated-JAK kinases and growth factor receptor kinases (Irie-Sasaki et al., 2001
), our preliminary data suggest an effect of SFK on inhibitory control of growth factor receptors.
Strikingly, in mice of a mixed genetic background (B6, Balb/c and 129) the B cell developmental block in the BM and the myeloproliferative syndrome were much delayed. Genetic modifiers may contribute to the severity of the disease. The discordant phenotypes also suggest that the early B cell developmental block in the DKO mice is not B-cell autonomous. A number of cytokines produced by myeloid cells such as type I interferons can suppress B cell development (Lin et al., 1998
). Relatively normal B-cell development in the BM of the young mixed lineage mice suggests that lack of CD148 and CD45 per se
is not sufficient to block very early B cell development and that other factors contribute to the phenotype as the disease progresses. In addition, data from our competitively reconstituted chimeras more clearly demonstrated that the very early B cell developmental block seen in B6 mice is secondary to some other not fully understood effects, possibly coming from a dysregulated myeloid compartment. In fact, the only B cell developmental block that is most likely intrinsic was observed during immature to mature B cell transition, given the discordant results obtained between wild-type and DKO B cells in healthy chimeric mice at week 6 post transplantation. It is not clear how much of this developmental defect is due to the CD45 deficiency alone. Further clarification of this issue will require B cell lineage-specific conditional DKO mice.
At the molecular level, we observed clear BCR signaling defects in DKO peripheral B cells. In the absence of CD45 and CD148, the cells maintained a high phosphorylation level of the C-terminal tyrosine of Lyn, thus keeping the kinase in the inactive conformation. As a likely consequence, the DKO B cells had impaired calcium flux following BCR ligation and failed to activate PLCγ2 and ERK. Importantly, the same defects in calcium flux and ERK activation in the DKO B cells occurred in competitively reconstituted chimeras, suggesting that the BCR signaling defects are in fact B cell intrinsic. Moreover, the signaling defects observed in the in vitro
expanded BMDMs were very similar to the defects observed in B cells. Finally, our results from DKO B cells are also consistent with mutants of the DT40 chicken B-cell line, which does not express endogenous CD148 (J.L. unpublished observation). CD45 deficient and Lyn deficient DT40 lines share very similar BCR-induced calcium signaling defects to our DKO B cells, exhibiting delayed and decreased peak responses (Takata et al., 1994
; Yanagi et al., 1996
). In contrast, calcium flux in Syk deficient DT40 cells is completely abolished (Takata et al., 1994
). These data suggest that via the regulation of SFKs, CD148 and CD45 contribute to the early peak calcium flux following BCR stimulation, whereas Syk may be responsible for sustaining the calcium flux after receptor clustering. A similar model may hold for the partial defects seen in the FcR-mediated signaling in macrophages. It is also important to stress that most of the signaling defects we observed in both B cells and macrophages were independent of the genetic background.
Although our study demonstrates that in B cells and macrophages CD148 and CD45 have similar overlapping functions, it is not clear whether there is any unique role for CD148 or CD45 in these lineages. Their very distinct structures suggest that they may be subject to different regulatory mechanisms. Previously, CD148 was considered to have an inhibitory role. In non-hematopoietic cells, CD148 has been reported to act as a tumor suppressor and inhibit proliferation of many different cell types predominantly of epithelial origin, most likely via dephosphorylation of different growth factor receptors (Balavenkatraman et al., 2006
; Jandt et al., 2003
; Keane et al., 1996
; Kovalenko et al., 2000
; Lesueur et al., 2005
; Palka et al., 2003
; Ruivenkamp et al., 2003
; Ruivenkamp et al., 2002
; Trapasso et al., 2000
; Trapasso et al., 2004
). It is possible that CD148 plays different roles depending on cell type and redundancy with other proteins. The overall phenotype reflects a net effect on the entire spectrum of available substrates that may be different in distinct cell types. It is possible that in B cells and myeloid cells the positive regulatory role of CD148 on Src-kinases prevails. Indeed, a study on rat thyroid carcinoma showed that overexpression of CD148 led to specific dephosphorylation of the C-terminal regulatory phosphotyrosine of c-Src that led to increased substratum adhesion (Pera et al., 2005
Our study identifies CD148 as an important regulator of SFKs and suggests that it does so in concert with CD45 in at least some hematopoietic lineages. In B cells and macrophages, CD148 dephosphorylates the negative regulatory phosphotyrosine of SFKs and therefore together with CD45 helps to set the threshold for SFK activation and, hence, the threshold for the immunoreceptor signaling in these cells. Since SFKs are important components of many other signaling pathways in hematopoietic cells, it is reasonable to expect that CD148 may have complex effects at multiple levels of immune cell regulation. Future studies of CD148 may provide not only deeper insights into the function of this RPTP itself but also of SFKs and immune cell regulation, in general.