CD98 Heavy Chain Research Articles

Abstract 3881: The expression of CD98hc might be a marker of radiation resistance in HNSCC

Abstract Introduction: Head and Neck squamous cell carcinoma(HNSCC) represents an aggressive malignancy. Although new advanced therapeutic strategies have been applied to date, the 5-year survival rate have not been considerably improved. Generally speaking, high radio-sensitive HNSCC has good prognosis, however, there are radio-resistant HNSCC. Recently, Sanne et al, have reported CD98 is one of the marker of Cancer Stem Cell. Moreover, the over expression of CD98hc is high associated with different cancers including the colon cancer, renal cancer. CD98 is heterodimeric protein that comprises a heavy chain and a light chain. The CD98 heavy chain is type 2 linkage to one of 6 amino acid transporters. We hypothesize that irradiation may contribute to overexpression of CD98hc and expansion Cancer Stem Cell population in HNSCC. Methods:cell culture: Ho-1-u-1(Mouth floor), Sa3(Gingiva), HSC2, HSC3,HSC4(tongue) were used for this experiment. These cell lines were exposed to 60 Gy (2Gy/day) to establish stable cell lines to radiation.Flow cytometry: For analysis of CD98-positive fraction, cell pellets were incubated with FITC-conjugated anti-human CD98hc mAb at the dilution of 1:11 at 4°C for 15min.Sphere formation assay: Cells were cultured in a serum-free medium. 1x104 cells were seeded in 80μl of serum free RPMI1640 medium. The recipe of which was mentioned above. Containing 1.0% agar on 100μl of a solidified serum free RPMI1640 was poured on the top of each well. The cells were incubated for 20 days in the same atomosphere as that for the monolayer culture.Migration Assay: Radius 24 well Cell Migration (Cell Biolabs, INC.) were purchased. The experiment was conducted according to the decided plotocol. Result: In radiation resistant cells, spindle-shaped and large cytoplasmic cells were founded. After irradiation, the expression of CD98hc increased in all cell lines. In Ho-1-u-1, the population was divided into two distinct subpopulations showing a higher and a lower expression of CD98hc. Irradiated cancer cells exhibit an efficient ability to developed numerous large sphere. Irradiation had enhanced self-renewal of Cancer Stem Cells. Cell motility was higher in radio-resistant cells than their parental cells. Conclusion: Radio-resistant cells overexpressed CD98hc. In other words, CD98hc may be a marker of radiation resistance. Since incomplete radiation therapy can expand Cancer Stem Cell population, so elucidation of mechanism of radiation resistance will improved radiotherapy. CD98hc might be the target for more efficient treatment. Citation Format: Yohei Kawasaki, Yasufumi Omori. The expression of CD98hc might be a marker of radiation resistance in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3881. doi:10.1158/1538-7445.AM2017-3881

Herpes Simplex Virus 1 UL34 Protein Regulates the Global Architecture of the Endoplasmic Reticulum in Infected Cells.

Upon herpes simplex virus 1 (HSV-1) infection, the CD98 heavy chain (CD98hc) is redistributed around the nuclear membrane (NM), where it promotes viral de-envelopment during the nuclear egress of nucleocapsids. In this study, we attempted to identify the factor(s) involved in CD98hc accumulation and demonstrated the following: (i) the null mutation of HSV-1 UL34 caused specific dispersion throughout the cytoplasm of CD98hc and the HSV-1 de-envelopment regulators, glycoproteins B and H (gB and gH); (ii) as observed with CD98hc, gB, and gH, wild-type HSV-1 infection caused redistribution of the endoplasmic reticulum (ER) markers calnexin and ERp57 around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of these markers; (iii) the ER markers colocalized efficiently with CD98hc, gB, and gH in the presence and absence of UL34 in HSV-1-infected cells; (iv) at the ultrastructural level, wild-type HSV-1 infection caused ER compression around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of the ER; and (v) the UL34-null mutation significantly decreased the colocalization efficiency of lamin protein markers of the NM with CD98hc and gB. Collectively, these results indicate that HSV-1 infection causes redistribution of the ER around the NM, with resulting accumulation of ER-associated CD98hc, gB, and gH around the NM and that UL34 is required for ER redistribution, as well as for efficient recruitment to the NM of the ER-associated de-envelopment factors. Our study suggests that HSV-1 induces remodeling of the global ER architecture for recruitment of regulators mediating viral nuclear egress to the NM.IMPORTANCE The ER is an important cellular organelle that exists as a complex network extending throughout the cytoplasm. Although viruses often remodel the ER to facilitate viral replication, information on the effects of herpesvirus infections on ER morphological integrity is limited. Here, we showed that HSV-1 infection led to compression of the global ER architecture around the NM, resulting in accumulation of ER-associated regulators associated with nuclear egress of HSV-1 nucleocapsids. We also identified HSV-1 UL34 as a viral factor that mediated ER remodeling. Furthermore, we demonstrated that UL34 was required for efficient targeting of these regulators to the NM. To our knowledge, this is the first report showing that a herpesvirus remodels ER global architecture. Our study also provides insight into the mechanism by which the regulators for HSV-1 nuclear egress are recruited to the NM, where this viral event occurs.

CD98 regulates vascular smooth muscle cell proliferation in atherosclerosis

Background and aimsVascular smooth muscle cells (VSMC) migrate and proliferate to form a stabilizing fibrous cap that encapsulates atherosclerotic plaques. CD98 is a transmembrane protein made of two subunits, CD98 heavy chain (CD98hc) and one of six light chains, and is known to be involved in cell proliferation and survival. Because the influence of CD98hc on atherosclerosis development is unknown, our aim was to determine if CD98hc expressed on VSMC plays a role in shaping the morphology of atherosclerotic plaques by regulating VSMC function. MethodsIn addition to determining the role of CD98hc in VSMC proliferation and apoptosis, we utilized mice with SMC-specific deletion of CD98hc (CD98hcfl/flSM22αCre+) to determine the effects of CD98hc deficiency on VSMC function in atherosclerotic plaque. ResultsAfter culturing for 5 days in vitro, CD98hc−/− VSMC displayed dramatically reduced cell counts, reduced proliferation, as well as reduced migration compared to control VSMC. Analysis of aortic VSCM after 8 weeks of HFD showed a reduction in CD98hc−/− VSMC proliferation as well as increased apoptosis compared to controls. A long-term atherosclerosis study using SMC-CD98hc−/−/ldlr−/− mice was performed. Although total plaque area was unchanged, CD98hc−/− mice showed reduced presence of VSMC within the plaque (2.1 ± 0.4% vs. 4.3 ± 0.4% SM22α-positive area per plaque area, p < 0.05), decreased collagen content, as well as increased necrotic core area (25.8 ± 1.9% vs. 10.9 ± 1.6%, p < 0.05) compared to control ldlr−/− mice. ConclusionsWe conclude that CD98hc is required for VSMC proliferation, and that its deficiency leads to significantly reduced presence of VSMC in the neointima. Thus, CD98hc expression in VSMC contributes to the formation of plaques that are morphologically more stable, and thereby protects against atherothrombosis.

Select membrane proteins modulate MNV-1 infection of macrophages and dendritic cells in a cell type-specific manner

Noroviruses cause gastroenteritis in humans and other animals, are shed in the feces, and spread through the fecal-oral route. Host cellular expression of attachment and entry receptors for noroviruses is thought to be a key determinant of cell tropism and the strict species-specificity. However, to date, only carbohydrates have been identified as attachment receptors for noroviruses. Thus, we investigated whether host cellular proteins play a role during the early steps of norovirus infection. We used murine norovirus (MNV) as a representative norovirus, since MNV grows well in tissue culture and is a frequently used model to study basic aspects of norovirus biology. Virus overlay protein binding assay followed by tandem mass spectrometry analysis was performed in two permissive cell lines, RAW264.7 (murine macrophages) and SRDC (murine dendritic cells) to identify four cellular membrane proteins as candidates. Loss-of-function studies revealed that CD36 and CD44 promoted MNV-1 binding to primary dendritic cells, while CD98 heavy chain (CD98) and transferrin receptor 1 (TfRc) facilitated MNV-1 binding to RAW 264.7 cells. Furthermore, the VP1 protruding domain of MNV-1 interacted directly with the extracellular domains of recombinant murine CD36, CD98 and TfRc by ELISA. Additionally, MNV-1 infection of RAW 264.7 cells was enhanced by soluble rCD98 extracellular domain. These studies demonstrate that multiple membrane proteins can promote efficient MNV-1 infection in a cell type-specific manner. Future studies are needed to determine the molecular mechanisms by which each of these proteins affect the MNV-1 infectious cycle.

Impaired CD98 signaling protects against graft-versus-host disease by increasing regulatory T cells

Graft-versus-host disease (GvHD) is a major barrier to the broader use of allogenic hematopoietic stem cell transplantation for non-malignant clinical applications. A murine model of C57BL/6 to B6D2F1 acute GvHD was employed with T lymphocytes harboring a deletion of the CD98 heavy chain (CD98hc−/−) as donor cells. The CD98hc−/− resulted in lower responses to alloantigen stimulation in a mixed leukocyte reaction assay, and prevented the mortality associated with disease progression. The percentage of donor CD8 T lymphocytes was significantly decreased, while the percentage of Foxp3-positive regulatory T cells (Tregs) in recipients was increased by CD98hc−/−. Decreased expression of FAS, FASL, ICOS, ICOSL, PD-1 and PD-L1 by donor CD8 T cells, and mRNA expression of cytotoxic T cell-related cytokines in the recipients were shown in those with CD98hc−/−. Fewer infiltrated cells are found in the lungs, liver, tongue and skin of recipients with CD98hc−/− compared with the wild type recipients. Taken together, our data indicate that T cell-specific deletion of CD98hc can contribute to the prevention of GvHD development due to the attenuation of lymphocyte migration and by increasing the generation of Treg cells. These findings are expected to make it possible to develop novel approaches for the prevention of GvHD.

Conditional deletion of CD98hc inhibits osteoclast development

The CD98 heavy chain (CD98hc) regulates virus-induced cell fusion and monocyte fusion, and is involved in amino acid transportation. Here, we examined the role that CD98hc plays in the formation of osteoclasts using CD98hcflox/floxLysM-cre peritoneal macrophages (CD98hc-defect macrophages). Peritoneal macrophages were stimulated with co-cultured with osteoblasts in the presence of 1,25(OH)2 vitamin D3, and thereafter stained with tartrate-resistant acid phosphatase staining solution. The multinucleated osteoclast formation was severely impaired in the peritoneal macrophages isolated from the CD98hc-defect mice compared with those from wild-type mice. CD98hc mediates integrin signaling and amino acid transport through the CD98 light chain (CD98lc). In integrin signaling, suppression of the M-CSF-RANKL-induced phosphorylation of ERK, Akt, JNK and p130Cas were observed at the triggering phase in the CD98h-defect peritoneal macrophages. Moreover, we showed that the general control non-derepressible (GCN) pathway, which was activated by amino acid starvation, was induced by the CD98hc-defect peritoneal macrophages stimulated with RANKL. These results indicate that CD98 plays two important roles in osteoclast formation through integrin signaling and amino acid transport.

CD98 Heavy Chain Is a Potent Positive Regulator of CD4+ T Cell Proliferation and Interferon-γ Production In Vivo.

Upon their recognition of antigens presented by the MHC, T cell proliferation is vital for clonal expansion and the acquisition of effector functions, which are essential for mounting adaptive immune responses. The CD98 heavy chain (CD98hc, Slc3a2) plays a crucial role in the proliferation of both CD4+ and CD8+ T cells, although it is unclear if CD98hc directly regulates the T cell effector functions that are not linked with T cell proliferation in vivo. Here, we demonstrate that CD98hc is required for both CD4+ T cell proliferation and Th1 functional differentiation. T cell-specific deletion of CD98hc did not affect T cell development in the thymus. CD98hc-deficient CD4+ T cells proliferated in vivo more slowly as compared with control T cells. C57BL/6 mice lacking CD98hc in their CD4+ T cells could not control Leishmania major infections due to lowered IFN-γ production, even with massive CD4+ T cell proliferation. CD98hc-deficient CD4+ T cells exhibited lower IFN-γ production compared with wild-type T cells, even when comparing IFN-γ expression in cells that underwent the same number of cell divisions. Therefore, these data indicate that CD98hc is required for CD4+ T cell expansion and functional Th1 differentiation in vivo, and suggest that CD98hc might be a good target for treating Th1-mediated immune disorders.

Transport of Iodothyronines by Human L-Type Amino Acid Transporters.

Thyroid hormone (TH) transporters facilitate cellular TH influx and efflux, which is paramount for normal physiology. The L-type amino acid transporters LAT1 and LAT2 are known to facilitate TH transport. However, the role of LAT3, LAT4, and LAT5 is still unclear. Therefore, the aim of this study was to further characterize TH transport by LAT1 and LAT2 and to explore possible TH transport by LAT3, LAT4, and LAT5. FLAG-LAT1-5 constructs were transiently expressed in COS1 cells. LAT1 and LAT2 were cotransfected with the CD98 heavy chain. Cellular transport was measured using 10 nM (125)I-labeled T4, T3, rT3, 3,3'-T2, and 10 μM [(125)I]3'-iodotyrosine (MIT) as substrates. Intracellular metabolism of these substrates was determined in cells cotransfected with either of the LATs with type 1 or type 3 deiodinase. LAT1 facilitated cellular uptake of all substrates and LAT2 showed a net uptake of T3, 3,3'-T2, and MIT. Expression of LAT3 or LAT4 did not affect transport of T4 and T3 but resulted in the decreased cellular accumulation of 3,3'-T2 and MIT. LAT5 did not facilitate the transport of any substrate. Cotransfection with LAT3 or LAT4 strongly diminished the cellular accumulation of 3,3'-T2 and MIT by LAT1 and LAT2. These data were confirmed by metabolism studies. LAT1 and LAT2 show distinct preferences for the uptake of the different iodocompounds, whereas LAT3 and LAT4 specifically facilitate the 3,3'-T2 and MIT efflux. Together our findings suggest that different sets of transporters with specific influx or efflux capacities may cooperate to regulate the cellular thyroid state.

Herpes Simplex Virus 1 Recruits CD98 Heavy Chain and β1 Integrin to the Nuclear Membrane for Viral De-Envelopment.

Herpesviruses have evolved a unique mechanism for nucleocytoplasmic transport of nascent nucleocapsids: the nucleocapsids bud through the inner nuclear membrane (INM; primary envelopment), and the enveloped nucleocapsids then fuse with the outer nuclear membrane (de-envelopment). Little is known about the molecular mechanism of herpesviral de-envelopment. We show here that the knockdown of both CD98 heavy chain (CD98hc) and its binding partner β1 integrin induced membranous structures containing enveloped herpes simplex virus 1 (HSV-1) virions that are invaginations of the INM into the nucleoplasm and induced aberrant accumulation of enveloped virions in the perinuclear space and in the invagination structures. These effects were similar to those of the previously reported mutation(s) in HSV-1 proteins gB, gH, UL31, and/or Us3, which were shown here to form a complex(es) with CD98hc in HSV-1-infected cells. These results suggested that cellular proteins CD98hc and β1 integrin synergistically or independently regulated HSV-1 de-envelopment, probably by interacting directly and/or indirectly with these HSV-1 proteins. Certain cellular and viral macromolecular complexes, such as Drosophila large ribonucleoprotein complexes and herpesvirus nucleocapsids, utilize a unique vesicle-mediated nucleocytoplasmic transport: the complexes acquire primary envelopes by budding through the inner nuclear membrane into the space between the inner and outer nuclear membranes (primary envelopment), and the enveloped complexes then fuse with the outer nuclear membrane to release de-enveloped complexes into the cytoplasm (de-envelopment). However, there is a lack of information on the molecular mechanism of de-envelopment fusion. We report here that HSV-1 recruited cellular fusion regulatory proteins CD98hc and β1 integrin to the nuclear membrane for viral de-envelopment fusion. This is the first report of cellular proteins required for efficient de-envelopment of macromolecular complexes during their nuclear egress.

CD147-CD98hc complex contributes to poor prognosis of non-small cell lung cancer patients through promoting cell proliferation via the PI3K/Akt signaling pathway.

It has been reported that CD147 and CD98 heavy chain (CD98hc) form a complex on the cell plasma membrane of several cancers; however, whether this complex exists in non-small cell lung cancer (NSCLC) cells and affects the prognosis of patients remains to be elucidated. The expression of CD147 and CD98hc was assessed in tissue samples from 241 NSCLC patients and NSCLC cell lines. The correlation between CD147 and CD98hc expression and their association with the prognosis of NSCLC patients were analyzed. We also evaluated the impact of CD147 and CD98hc on the growth of NSCLC cells as well as Akt phosphorylation. Both CD147 and CD98hc were significantly upregulated in NSCLC cells, and their expression levels were significantly correlated (p < 0.001). Immunoflurenece staining and co-immunoprecipitation demonstrated that CD147 and CD98hc could form a complex on NSCLC cells. Compared with NSCLC patients with CD147-/CD98hc-, those with CD147+/CD98hc+ exhibited a significantly poor overall survival (OS) with a hazard ratio (HR) of 1.92 (p = 0.010), and a significantly increased risk of recurrence with a HR of 1.97 (p = 0.004). Also, we demonstrated that the proliferation of lung cancer cell lines was significantly affected by knockdown and force-expression of the CD147-CD98hc complex. Western blot analysis indicated that the phosphorylation of Akt in NSCLC cells was significantly affected by knockdown and overexpression of either or both CD147 and CD98hc. Our findings indicate that the CD147-CD98hc complex significantly contributes to poor prognosis of NSCLC patients through promoting cell proliferation via the PI3K/Akt pathway.

Calcium Ionophore A23187 Inhibits Human Parainfluenza Virus Type 2 Growth and Monoclonal Antibody against CD98 Heavy Chain Recovers the Inhibition

Calcium ionophore A23187 (A23187) inhibited human parainfluenza virus type 2 (hPIV-2) replication in LLCMK2 cells and the inhibition was almost completely recovered by monoclonal antibody (mAb) against CD98 heavy chain (CD98HC). A23187 considerably reduced the number of viruses released from the cells. Virus nucleoprotein (NP), fusion (F) and hemaggulutinin-neuraminidase (HN) gene syntheses were not inhibited. However, a direct immunofluorescence study showed that A23187 largely inhibited virus NP, F and HN protein syntheses. Using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix (M) protein (rghPIV-2i„M), it was found that virus entry into the cells was not inhibited, and that cell-to-cell spreading of virus was not blocked by A23187 either. The effects of A23187 on both actin microfilaments and microtubules were observed, and it was found to cause disruption of both of them. The effect of CD98HC mAb on the inhibition induced by A23187 was analyzed. CD98HC mAb recovered the inhibition by A23187. Virus production was considerably recovered. Virus protein was synthesized by the addition of CD98HC mAb. Almost normal actin microfilaments and microtubules were observed in the presence of CD98HC mAb. These results suggested that the inhibitory effect of A23187 was mainly caused by the inhibition of virus protein synthesis and disruption of cytoskeleton. The precise mechanisms of the inhibitory effect of A23187 and recovery from A23187-induced inhibition by CD98HC mAb are unclear, but the modulation of cytosolic Ca2+ concentration may be important for viral protein synthesis and for the preservation of cytoskeleton.

CD98hc regulates the development of experimental colitis by controlling effector and regulatory CD4+ T cells

CD4+ T cell activation is controlled by signaling through the T cell receptor in addition to various co-receptors, and is also affected by their interactions with effector and regulatory T cells in the microenvironment. Inflammatory bowel diseases (IBD) are caused by the persistent activation and expansion of auto-aggressive CD4+ T cells that attack intestinal epithelial cells. However, the molecular basis for the persistent activation of CD4+ T cells in IBD remains unclear. In this study, we investigated how the CD98 heavy chain (CD98hc, Slc3a2) affected the development of colitis in an experimental animal model. Transferring CD98hc-deficient CD4+CD25− T cells into Rag2−/− mice did not cause colitis accompanied by increasing Foxp3+ inducible regulatory T cells. By comparison, CD98hc-deficient naturally occurring regulatory T cells (nTregs) had a decreased capability to suppress colitis induced by CD4+CD25− T cells, although CD98hc-deficient mice did not have a defect in the development of nTregs. Blocking CD98hc with an anti-CD98 blocking antibody prevented the development of colitis. Our results indicate that CD98hc regulates the expansion of autoimmune CD4+ T cells in addition to controlling nTregs functions, which suggests the CD98hc as an important target molecule for establishing strategies for treating colitis.

CD98 Increases Renal Epithelial Cell Proliferation by Activating MAPKs

CD98 heavy chain (CD98hc) is a multifunctional transmembrane spanning scaffolding protein whose extracellular domain binds with light chain amino acid transporters (Lats) to form the heterodimeric amino acid transporters (HATs). It also interacts with β1 and β3 integrins by its transmembrane and cytoplasmic domains. This interaction is proposed to be the mechanism whereby CD98 mediates cell survival and growth via currently undefined signaling pathways. In this study, we determined whether the critical function of CD98-dependent amino acid transport also plays a role in cell proliferation and defined the signaling pathways that mediate CD98-dependent proliferation of murine renal inner medullary collecting duct (IMCD) cells. We demonstrate that downregulating CD98hc expression resulted in IMCD cell death. Utilizing overexpression studies of CD98hc mutants that either lacked a cytoplasmic tail or were unable to bind to Lats we showed that CD98 increases serum-dependent cell proliferation by a mechanism that requires the CD98hc cytoplasmic tail. We further demonstrated that CD98-dependent amino acid transport increased renal tubular epithelial cell proliferation by a mechanism that does not require the CD98hc cytoplasmic tail. Both these mechanisms of increased renal tubular epithelial cell proliferation are mediated by Erk and p38 MAPK signaling. Although increased amino transport markedly activated mTor signaling, this pathway did not alter cell proliferation. Thus, these studies demonstrate that in IMCD cells, the cytoplasmic and extracellular domains of CD98hc regulate cell proliferation by distinct mechanisms that are mediated by common MAPK signaling pathways.

Deletion of CD98 Heavy Chain in T Cells Results in Cardiac Allograft Acceptance by Increasing Regulatory T Cells

Little is known about the CD98 heavy chain (CD98hc) in the T lymphocyte-mediated immune response to alloantigen. We used an in vitro mixed leukocyte reaction assay and a cardiac transplantation model to evaluate the mechanisms of CD98hc in regulating alloimmune responses. A T cell-specific deficiency of CD98hc resulted in lower responses to alloantigen stimulation in a mixed leukocyte reaction assay, and CD98hc-deficient mice accepted full major histocompatibility complex-mismatched cardiac allografts. Consistent with graft survival, the infiltration of the graft by immune cells in CD98hc-deficient mice was significantly lower than that in wild-type mice. A chemotaxis assay revealed the migration of CD98hc-deficient lymphocytes to decrease in the presence of CCL5 compared with wild-type cells. Moreover, the proportion of CD4/Foxp3-positive cells and Foxp3 messenger RNA increased significantly in CD98hc-deficient recipients, consistent with the down-regulation of mammalian target of rapamycin and PS6 kinase; and allograft permanent acceptance was shortened by the depletion of antibody-induced regulatory T cells. Finally, neutralizing antibody against CD98hc prolonged the cardiac allograft survival. Taken together, our data indicate that T cell-specific deficiency in CD98hc can contribute to cardiac allograft permanent acceptance correlating with the attenuation of lymphocyte migration and by increasing the generation of regulatory T cells. These findings are expected to make it possible to develop novel approaches for treating allograft rejection and promoting transplantation tolerance.

NIH3T3 cells overexpressing CD98 heavy chain resist early G1 arrest and apoptosis induced by serum starvation

CD98 is a heterodimeric glycoprotein of 125-kDa, which consists of a 90-kDa heavy chain (hc) subunit and 35-kDa to 55-kDa light chain (lc) subunits. It is strongly expressed on the surface of proliferating normal cells and almost all tumor cells. To investigate the participation of CD98 in cellular proliferation and malignant transformation, we analyzed cell-cycle progression of NIH3T3 clones transfected with cDNA of human CD98hc. Although NIH3T3 and control transfectant cells grown to the subconfluent state were arrested in the G0/G1 phase by serum starvation, considerable portions of CD98hc-transfected cells resided at S and G2/M phases. Under serum-starved and confluent conditions, significant fractions (20-25%) of NIH3T3 and control transfectant cells contained less than 2n content DNA, indicating occurrence of apoptosis, whereas no apoptotic cells were detected in CD98hc-transfectant cells. Under serum-starved conditions, a marked increase in the levels of cyclin D1 and cyclin E and a decrease in p16 were observed in CD98hc-transfectant cells. The reverse was true for NIH3T3 and control transfectant cells. Our results suggest that resistance to G1 arrest and apoptosis by CD98 overexpression are associated with high G1-cyclins and low p16 levels.

Loss of T cell CD98hc specifically ablates T cell clonal expansion and protects from autoimmunity. (102.16)

Abstract CD98hc (CD98 heavy chain, 4F2 antigen, Slc3a2) is a lymphocyte activation antigen with unclear function in T cells. Deletion of CD98hc in B cells leads to complete failure of B cell proliferation, plasma cell formation, and antibody secretion. Here we examined the role of T cell CD98 in protective and auto- immune responses by specifically deleting it in mouse T cells. Deletion of T cell CD98 prevented experimental autoimmune diabetes associated with reduced T cell clonal expansion. However, initial T cell homing to pancreatic islets was unimpaired. In contrast to B cells, CD98-null T cells showed only moderately impaired antigen-driven proliferation in vitro and nearly normal homeostatic proliferation. Furthermore, these cells were activated by antigen and produced cytokines (CD4) and efficiently killed targets (CD8). The integrin binding domain of CD98hc was necessary and sufficient for full clonal expansion, highlighting the role of adhesive signaling in T cell proliferation and autoimmune disease. When CD98-null T cells were pre-expanded in vitro, they adoptively transferred diabetes, establishing that impaired clonal expansion was responsible for protection from disease. The integrin binding domain of CD98hc is thus required for antigen-driven T cell clonal expansion in the pathogenesis of an autoimmune disease and may represent a useful therapeutic target.

Extracellular domain of CD98hc is required for early murine development

BackgroundThe multifunctional protein CD98 heavy chain (CD98hc, Slc3a2) associates with integrin β1 through its cytoplasmic and transmembrane domains and the CD98hc-mediated integrin signaling is required for maintenance of ES cell proliferation. CD98hc-null mice exhibit early post-implantation lethality similar to integrin β1-null mice, supporting the importance of its interaction with integrin β1. On the other hand, the extracellular domain of CD98hc interacts with L-type amino acid transporters (LATs) and is essential for appropriate cell surface distribution of LATs. LATs mediate the transport of amino acids and other molecules such as thyroid hormone. In this respect, CD98hc may also affect development via these transporters.ResultsIn this study, mice were generated from embryonic stem (ES) cell line (PST080) harboring a mutant CD98hc allele (CD98hcΔ/+). Expression of the CD98hc mutant allele results in ΔCD98hc-β geo fusion protein where extracellular C-terminal 102 amino acids of CD98hc are replaced with β geo. Analyses of PST080 ES cells as well as reconstituted frog oocytes demonstrated that ΔCD98hc-β geo fusion protein preserved its ability to interact with integrin β1 although this mutant protein was hardly localized on the cell surface. These findings suggest that ΔCD98hc-β geo protein can mediate integrin signaling but cannot support amino acid transport through LATs. CD98hcΔ/+ mice were normal. Although some of the implantation sites lacked embryonic component at E9.5, all the implantation sites contained embryonic component at E7.5. Thus, CD98hcΔ/Δ embryos are likely to die between E7.5 and E9.5.ConclusionsConsidering that CD98hc complete knockout (CD98hc-/-) embryos are reported to die shortly after implantation, our findings suggest potential stage-specific roles of CD98hc in murine embryonic development. CD98hc may be essential for early post-implantation development by regulating integrin-dependent signaling, while the other function of CD98hc as a component of amino acid transporters may be required for embryonic development at later stages.

Dependence of proliferative vascular smooth muscle cells on CD98hc (4F2hc, SLC3A2)

Activation of vascular smooth muscle cells (VSMCs) to migrate and proliferate is essential for the formation of intimal hyperplasia. Hence, selectively targeting activated VSMCs is a potential strategy against vaso-occlusive disorders such as in-stent restenosis, vein-graft stenosis, and transplant vasculopathy. We show that CD98 heavy chain (CD98hc) is markedly up-regulated in neointimal and cultured VSMCs, and that activated but not quiescent VSMCs require CD98hc for survival. CD98hc mediates integrin signaling and localizes amino acid transporters to the plasma membrane. SMC-specific deletion of CD98hc did not affect normal vessel morphology, indicating that CD98hc was not required for the maintenance of resident quiescent VSMCs; however, CD98hc deletion reduced intimal hyperplasia after arterial injury. Ex vivo and in vitro, loss of CD98hc suppressed proliferation and induced apoptosis in VSMCs. Furthermore, reconstitution with CD98hc mutants showed that CD98hc interaction with integrins was necessary for the survival of VSMCs. These studies establish the importance of CD98hc in VSMC proliferation and survival. Furthermore, loss of CD98hc was selectively deleterious to activated VSMCs while sparing resident quiescent VSMCs, suggesting that activated VSMCs are physiologically dependent on CD98hc, and hence, CD98hc is a potential therapeutic target in vaso-occlusive disorders.

Drosophila expresses a CD98 transporter with an evolutionarily conserved structure and amino acid-transport properties

Mammalian CD98 heterodimeric amino acid transporters consist of a promiscuous single-pass transmembrane glycoprotein, CD98hc (CD98 heavy chain), and one of six multipass transmembrane proteins or 'light chains'. The heterodimeric complexes of CD98hc and the light chains LAT1 (L-type amino acid transporter 1) or LAT2 specifically promote sodium-independent System L exchange of neutral amino acids, including leucine. CD98hc is also implicated in other processes, including cell fusion, cell adhesion and activation of TOR (target of rapamycin) signalling. Surprisingly, recent reports suggested that insects lack a membrane-bound CD98hc, but in the present study we show that Drosophila CG2791 encodes a functional CD98hc orthologue with conservation in intracellular, transmembrane and extracellular domains. We demonstrate by RNA-interference knockdown in Drosophila Schneider cells that CG2791 and two Drosophila homologues of the mammalian CD98 light chains, Mnd (Minidiscs) and JhI-21, are required for normal levels of System L transport. Furthermore, we show that System L activity is increased by methoprene, an analogue of the developmentally regulated endocrine hormone juvenile hormone, an effect that is potentially mediated by elevated Mnd expression. Co-expression of CG2791 and JhI-21, but not CG2791 and Mnd, in Xenopus oocytes mediates System L transport. Finally, mapping of conserved sequences on to the recently determined crystal structure of the human CD98hc extracellular domain highlights two conserved exposed hydrophobic patches at either end of the domain that are potential protein-protein-interaction surfaces. Therefore our results not only show that there is functional conservation of CD98hc System L transporters in flies, but also provide new insights into the structure, functions and regulation of heterodimeric amino acid transporters.

Analyses of Transgenic Mice that Have Human CD98 Heavy Chain Gene

Transgenic (Tg) mice, which carry human CD98 heavy chain (hCD98HC) gene, were made for the first time. Biochemical analyses of total protein, albumin, cholesterol, glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and alkaline phosphatase (ALP) in the sera were carried out. The expression of hCD98HC and murine CD98HC (mCD98HC) mRNA, and the tissue distribution of hCD98HC and mCD98HC were determined.The values of total protein, albumin, cholesterol, GOT, GPT and ALP of Tg mice were about the same as those of normal mice. The mCD98HC mRNA was highly expressed in all the tissues of normal mice examined except for skeletal muscle, where it was expressed rather slightly. hCD98HC mRNA of Tg mice was detected on cDNA prepared using oligo-dT primer, and the mRNA was clearly found in brain, kidney, skin, spleen, stomach, testis and thymus. However the mRNA was rather slight in liver, skeletal muscle and intestine, and almost nothing in heart. Immunofluorescence study of murine tissue sections using monoclonal antibody (mAb) directed against mCD98HC revealed that mCD98HC was clearly expressed in renal proximal tubules of kidney, follicular epitherium and keratinocyte of skin, and interstitial space including Leydig cell of testis. However, hCD98HC in Tg mice, determined by immunostaining using hCD98HC mAb, was not detected in any of the tissues examined.