Involvement of a novel zinc finger protein, MIZF, in transcriptional repression by interacting with a methyl-CpG-binding protein, MBD2.

MBD2, a methyl-CpG-binding protein, is a component of the MeCP1 histone deacetylase (HDAC) complex and plays a critical role in DNA methylation-mediated transcriptional repression. To understand the molecular basis of the methylation-associated repression, we attempted to identify MBD2-interacting proteins by a yeast two-hybrid system. Using MBD2 as bait, we isolated a novel zinc finger protein, referred to as MIZF. A direct interaction between MBD2 and MIZF was confirmed by in vitro binding assays and immunoprecipitation experiments. Four of seven zinc fingers present in the C-terminal region of MIZF are required for binding with MBD2. The MIZF mRNA is expressed in all human tissues and cell lines examined. The subcellular localization of MIZF is distinct from that of MBD2, although both proteins co-localize in some areas of the nuclei; MIZF localizes diffusely in the nucleoplasmic region, whereas MBD2 preferentially localizes in major satellites. A reporter assay demonstrated that MIZF significantly abrogates transcriptional activities. This repression is attenuated by an HDAC inhibitor, trichostatin A, and is completely dependent on the interaction with MBD2. These results suggest that MIZF is abundantly present in cells and functions as a negative regulator of transcription by binding to MBD2 and recruiting HDAC-containing complexes.

Involvement of ErbB-2 in rheumatoid synovial cell growth.

OBJECTIVE: The synovial tissue affected by rheumatoid arthritis (RA) is characterized by hyperproliferation of synovial cells. High amounts of epidermal growth factor (EGF) in the synovial fluid of RA patients contribute to the growth of rheumatoid synovial cells. To characterize the receptor for EGF in rheumatoid synovial cells, the expression and function of ErbB family members were examined. METHODS: Synovial tissues were obtained from surgical excisions. The expression of ErbB products was examined by immunohistochemistry and immunoblotting by using specific antibodies. Primary cultures were established from the surgical materials. Cell growth was measured using MTT. The levels and phosphorylation state of the ErbB-2 protein were analyzed by immunoprecipitation and immunoblotting. RESULTS: The expression of ErbB-2, but not other ErbB-related products, was detected in synovium with RA as compared with that with osteoarthritis (OA) and ligament injury. Growth of primary synovial cells with RA was inhibited by genistein, a tyrosine kinase inhibitor, and herceptin, a specific monoclonal antibody against ErbB-2. Herceptin showed a small effect on growth of primary synovial cells with OA. EGF stimulated the phosphorylation of ErbB-2 in primary synovial cells with RA. This EGF-stimulated phosphorylation was completely abrogated by genistein and herceptin. CONCLUSION: ErbB-2 is expressed in rheumatoid synovial cells and may function as the receptor for EGF. Our data suggest that mitotic signals from EGF family members are transduced by ErbB-2 in these cells. Inhibition of ErbB-2 may provide a new approach to the effective treatment for RA.

Wavelength-specific activation of MAP kinase family proteins by monochromatic UV irradiation.

The depletion of stratospheric ozone causes related increase in UV light below about 310 nm, which significantly affects biological and ecological systems. To understand the wavelength-specific effects of UV light, Molt4 cells (human T lymphoma cells) were irradiated with a series of monochromatic UV lights and the activities of three members of the mitogen-activated protein (MAP) kinase group were examined. Extracellular signal-regulated kinase was specifically activated within 1 min after UV irradiation in the range 320-360 nm. In contrast, P38 kinase was activated by 270-280 nm light with a peak at 1 min after irradiation. c-Jun N-terminal kinase activation was observed in a narrow range of UV light with a sharp peak at 280 nm occurring in 10 min. JNK translocated from the cytosol to the nucleus upon irradiation, while P38 remained in the cytosol even after UV irradiation. The activation of three MAP kinases was prevented by antioxidant reagents, suggesting that an oxidative signal initiates these responses. These results confirm that UV light affects various cellular functions through the activation of intracellular signaling systems including MAP kinase family proteins. However, the UV-induced activities of the separate MAP kinases show distinctly different dose, time and wavelength dependencies.

Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia.

Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1beta or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis.

Cutting edge: essential role of phospholipase C-gamma 2 in B cell development and function.

Cross-linking of the B cell Ag receptor (BCR) induces the tyrosine phosphorylation of multiple cellular substrates, including phospholipase C (PLC)-gamma 2, which is involved in the activation of the phosphatidylinositol pathway. To assess the importance of PLC-gamma 2 in murine lymphopoiesis, the PLC-gamma 2 gene was inducibly ablated by using IFN-regulated Cre recombinase. Mice with a neonatally induced loss of PLC-gamma 2 function displayed reduced numbers of mature conventional B cells and peritoneal B1 cells and defective responses in vitro to BCR stimulation and in vivo to immunization with thymus-independent type II Ags. In contrast, T cell development and TCR-mediated proliferation were normal. Taken together, PLC-gamma 2 is a critical component of BCR signaling pathways and is required to promote B cell development.

Morphological changes and detachment of adherent cells induced by p122, a GTPase-activating protein for Rho.

We recently cloned a novel signaling molecule, p122, that shows a GTPase-activating activity specific for Rho and the ability to enhance the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C delta1 in vitro. Here we analyzed the in vivo function of p122. Microinjection of the GTPase-activating domain of p122 suppressed the formation of stress fibers and focal adhesions induced by lysophosphatidic acid, suggesting a GTPase-activating activity for Rho as in in vitro. Transfection of p122 also induced the disassembly of stress fibers and the morphological rounding of various adherent cells. Analyses using deletion and point mutants demonstrated that the GTPase-activating domain of p122 is responsible for the morphological changes and detachment and that arginine residues at positions 668 and 710 and a lysine residue at position 706 in the GTPase-activating domain are essential. Using Fluo-3-based Ca2+ microscopy, we found that p122 evoked a rapid elevation of intracellular Ca2+ levels, suggesting that p122 stimulates the phosphatidylinositol 4, 5-bisphosphate-hydrolyzing activity of phospholipase C delta1. These results demonstrate that p122 synergistically functions as a GTPase-activating protein specific for Rho and an activator of phospholipase C delta1 in vivo and induces morphological changes and detachment through cytoskeletal reorganization.

Different growth properties in response to epidermal growth factor and interleukin-6 of primary keratinocytes derived from normal and psoriatic lesional skin.

Epidermal growth factor (EGF) family members and its receptor (EGFR) are thought to have an important role in the proliferation of epidermal keratinocytes. In this report, we investigated the EGF/EGFR system in primary keratinocytes derived from normal and psoriatic lesional skin. EGF elicited the growth of both normal human keratinocytes (NHKs) and psoriatic lesional keratinocytes (PLKs). Interleukin-6 (IL-6) potentiated the EGF-dependent growth of NHKs, but has no observable effect on PLKs, while IL-6 itself showed no growth-stimulating activities in both cell types. Immunodetection and in situ hybridization analyses revealed that IL-6 induces EGFR expression in NHKs in a time- and dose-dependent manner. This EGFR expression decreased reversibly to an undetectable level when IL-6-treated NHKs were re-cultured in IL-6-free conditions. On the other hand, PLKs expressed high levels of EGFR even when unstimulated and the expression level was not affected by IL-6 stimulation. These results suggest that the EGF/EGFR system is involved in the growth of NHKs and PLKs and that IL-6 potentiates NHK growth partly through the induction of EGFR. The different EGFR regulatory system may contribute to the pathogenesis of psoriasis.

Visualization of protein kinase activities in single cells by antibodies against phosphorylated vimentin and GFAP.

Vimentin and glial fibrillary acidic protein (GFAP) are intermediate filament proteins expressed in the cytoplasm of various types of cells. The head domains of these proteins are phosphorylated by various protein kinases. Site- and phosphorylation-specific antibodies which recognize a phosphorylated serine/threonine residue in the head domains and its flanking sequence provide a useful tool to monitor and visualize protein kinase activities in single cells.

Detection of protein kinase activity specifically activated at metaphase-anaphase transition.

We have previously reported that Ser13 and Ser34 on glial fibrillary acidic protein (GFAP) in the cleavage furrow of glioma cells are phosphorylated during late mitotic phase (Matsuoka, Y., K. Nishizawa, T. Yano, M. Shibata, S. Ando, T. Takahashi, and M. Inagaki. 1992, EMBO (Eur. Mol. Biol. Organ.) J. 11:2895-2902). This observation implies a possibility that there is a protein kinase specifically activated at metaphase-anaphase transition. To further analyze the cell cycle-dependent GFAP phosphorylation, we prepared monoclonal antibodies KT13 and KT34 which recognize the phosphorylation of GFAP at Ser13 and Ser34, respectively. Immunocytochemical studies with KT13 and KT34 revealed that the GFAP phosphorylation in the cleavage furrow during late mitotic phase occurred not only in glioma cells but also in human SW-13 and mouse Ltk- cells in which GFAP was ectopically expressed, thus the phosphorylation can be monitored in a wide range of cell types. Furthermore, we detected kinase activity which phosphorylates GFAP at Ser13 and Ser34 in the lysates of late mitotic cells but not in those of interphase cells or early mitotic cells. These results suggest that there exists a protein kinase which is specifically activated at the transition of metaphase to anaphase not only in GFAP-expressing cells but also in cells without GFAP.

Primary structure of light and heavy chain variable regions of antibodies recognizing phosphorylated vimentins.

We determined the primary structure of three types of monoclonal antibodies against phosphorylated vimentin, 4A4, YT33, and MO82, which recognize phosphorylated Ser55, Ser33, and Ser82 on vimentin, respectively. The amino acid sequences between these antibodies and the anti-phosphotyrosine antibodies previously reported, (Asn/Gln)-X-(Gln/Tyr)-Ser-Tyr in the complimentarity determining region (CDR) 3 of the light chain of 4A4 and YT33, Asn-Pro-Asn-(Asn/Ser)-Gly-X-(Ser/Thr)-(Ser/Thr)-Tyr-Asn-Gln-(Arg/Lys)-Ph e-Lys in the heavy chain CDR2 of MO82, and Lys-X-Ser-(Ser/Asn) in the heavy chain CDR3 of YT33 and MO82, were highly conserved. These motifs may play a role in recognizing phosphate groups of phosphoserine and phosphotyrosine.

Differential targeting of protein kinase C and CaM kinase II signalings to vimentin.

Hydrolysis of inositol phospholipids by receptor stimulation activates two separate signaling pathways, one leading to the activation of protein kinase C (C kinase) via formation of diacylglycerol. The other is the inositol trisphosphate (IP3)/Ca2+ pathway and a major downstream kinase which is activated is Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). To examine signaling pathways of C kinase and CaM kinase II to the cytoskeletal protein vimentin, we prepared monoclonal antibodies YT33 and MO82 which recognize the phosphorylation state of vimentin by C kinase and by CaM kinase II, respectively. Ectopic expression of constitutively active C kinase or CaM kinase II in primary cultured astrocytes by microinjection of the corresponding expression vectors induced phosphorylation of vimentin at each specific phosphorylation site, followed by reorganization of vimentin filament networks. In contrast, simultaneous activation of C kinase and CaM kinase II by inositol phospholipid hydrolysis with receptor stimulation led to an exclusive phosphorylation of vimentin at the CaM kinase II site, not at the site of C kinase. These results indicate that the intracellular targeting of C kinase and CaM kinase II signalings to vimentin is regulated separately, under physiological conditions.

The role of the conserved residue in pocket A and the polymorphic residue in pocket E of HLA-B*3501 in presentation of human minor histocompatibility peptides to T cells.

We investigated T cell recognition for human minor histocompatibility (hmH) peptides using HLA-B*3501 restricted, hmH specific cytotoxic T lymphocytes (CTL) clones. These CTL clones killed C1R cells expressing HLA-B*3501 but not C1R cells expressing chimeric antigens between HLA-B*3501 and HLA-B*5101. They also failed to kill C1R cells expressing HLA-B*3501 mutants at residue 152 (B*3501-V152E) or at residue 171 (B*3501-Y171H). The CTL clone failed to kill C1R cells expressing these mutant molecules loaded with the hmH peptides isolated from C1R-B*3501 cells although it killed a self-B cell line expressing HLA-B3501 loaded with the specific hmH peptides. The CTL clone also failed to kill T2 cells expressing the mutant molecules loaded with the specific peptides whereas it killed T2 cells expressing HLA-B*3501 loaded with the specific peptide. On the other hand, naturally occurring specific hmH peptides were isolated from purified B*3501-V152E and B*3501-Y171H molecules, indicating that both HLA-B*3501-V152E and HLA-B*3501-Y171H molecules can bind the hmH peptides. These findings indicate that both the conserved residue 171 in pocket A and the polymorphic residue 152 in pocket E are critical in recognition of the T cells but not binding of the hmH peptides. Furthermore, these results provide the possibility that the TCR recognizes a conformational structure of hmH peptides bound to HLA-B*3501 molecules.

Different effects of substitutions at residues 224 and 228 of MHC class I on the recognition of CD8.

Previous studies indicated that weak xenoresponse to HLA class I by mouse T cells is due to the inefficient interaction of mouse CD8 with the alpha 3 domain of HLA class I. The present study using chimeric H-2Kb molecules with recombinant alpha 3 domain between H-2Kb and HLA-B7 as well as single amino acid mutants of H-2Kb demonstrated that each substitution at residues 224 and 228 affects recognition of CD8-dependent mouse CTL clones. On the other hand, reactivity of IL-2-producing H-2Kb-specific T cell hybridoma transfected with mouse CD8 alpha was abrogated by substitution at residue 224 but not by that at residue 228. This indicates that the substitution at residue 228 affects recognition of CD8-dependent CTL but does not critically affect binding of CD8 to MHC class I molecules, although residue 224 abrogates binding of CD8. The model structure of the alpha 3 domain of H-2Kb suggests that the substitution at residue 224 induces conformational change of CD8 binding loop, whereas minimum structure change by the substitution at residue 228 is expected. It is therefore speculated that minimum structure change of CD8 binding loop by substitution at residue 228 may influence binding affinity of CD8, which abrogates recognition of CD8-dependent CTL but not IL-2 production of the CD8-dependent T cell hybridoma.

Expression of human minor histocompatibility antigen on cultured kidney cells.

Incompatibility of human minor histocompatibility (hmH) antigens can induce rejection of grafts in organ transplantation and graft-versus-host reactions in bone marrow transplantation. In spite of their importance in clinical transplantation, hmH antigens are not well studied. Previous studies have demonstrated the expression of hmH antigens on T and B cells, hematopoietic progenitor cells and keratinocytes. We have for the first time demonstrated the expression of hmH antigens on cultured kidney cells using HLA-B35-restricted, hmH antigen-specific cytotoxic T lymphocyte (CTL) clones, which were previously established from a patient who rejected two kidneys from HLA-identical sisters. The CTL clones could not kill cultured kidney cells. Since cultured kidney cells expressed very low levels of HLA class I antigens it was thought that their failure to be killed by the CTL clones was due to lack of expression of HLA-B35 antigens. After induction of class I antigens on cultured kidney cells by interferon-gamma (IFN-gamma), the IFN-gamma-treated cultured kidney cells were killed by the CTL clones. Furthermore, we isolated hmH antigens as peptides from cultured kidney cells after treatment with IFN-gamma. These results indicate that cultured kidney cells express hmH antigens when HLA class I antigen is induced by IFN-gamma and hmH antigens on cultured kidney cells are recognized by T cells as peptides presented by HLA-B35 molecules.

Structural and functional analysis of monomorphic determinants recognized by monoclonal antibodies reacting with the HLA class I alpha 3 domain.

To identify mAb reacting with the HLA class I alpha 3 domain, 14 mAb recognizing monomorphic determinants expressed on HLA-A, B, and C Ag or restricted to HLA-B Ag were screened in indirect immunofluorescence with mouse L cells expressing HLA-B7/H-2Kb chimeric Ag. mAb CR1S63, CR10-215, CR11-115, and W6/32 were found to react with the HLA class I alpha 3 domain in addition to the alpha 2 domain. mAb Q1/28 and TP25.99 were found to react only with the HLA class I alpha 3 domain. The determinants recognized by the six mAb were mapped on the HLA class I alpha 3 domain by indirect immunofluorescence staining of L cells expressing H-2Kb Ag containing different segments of the HLA-B7 alpha 3 domain chimerized with the H-2Kb alpha 3 domain. mAb TP25.99 reacts with chimeric Ag containing the HLA-B7 184 to 199 stretch, mAb CR10-215 and CR11-115 react with chimeric Ag containing the HLA-B7 184 to 246 stretch, mAb CR1S63 and Q1/28 react with chimeric Ag containing the HLA-B7 184 to 256 stretch, and mAb W6/32 reacts with chimeric Ag containing the whole HLA-B7 alpha 3 domain. Functional analysis using human CD8 alpha-bearing mouse H-2Kb-specific T cell hybridoma cells (HTB-Leu2) showed that only mAb TP25.99 inhibited IL-2 production by HTB-Leu2 cells stimulated with L cells expressing KbKbB7 Ag. This inhibition may occur because of the spatial proximity of the determinant defined by mAb TP25.99 to the CD8 alpha binding loop and/or because of change(s) in the conformation of the CD8 alpha binding loop induced by the binding of mAb TP25.99 to the HLA class I molecule. Furthermore, mAb TP25.99 inhibited the cytotoxicity of CD8-dependent and CD8-independent CTL clones. These results indicate that mAb TP25.99 has unique specificity and functional characteristics. Therefore it represents a valuable probe to characterize the role of the HLA class I alpha 3 domain in immunologic phenomena.

Isolation of human minor histocompatibility peptides.

Incompatibility of human minor histocompatibility (hmH) antigens induces rejection of grafts in organ transplantation and graft versus host disease in bone marrow transplantation if donor and recipient are matched for human leukocyte antigen (HLA) genes. These antigens are recognized only by T cells. We describe here the isolation of hmH peptides recognized by a hmH antigen specific, HLA-B35 restricted CTL clone which was derived from a patient who rejected the kidneys from two HLA-identical sisters. Naturally occurring hmH peptides were isolated from a donor derived B cell line and an HLA-B35 transfected human B cell line by acid elution. Analysis of various HLA class I transfectant cells demonstrated that MHC class I molecules themselves determine the peptides which are naturally processed and presented to T cells.

Detection of allogeneic Qa/TL and Ly specificities on murine tumor cells with IgD in tumor-regressor serum.

Serum from C3H/He mice, which show regression of MM2 tumor cells after transplantation and removal (regressor serum, RS) contains non-gammaglobulin components that cross-react with various tumor cells of mice [22, 23]. In addition to tumor cells, various allogeneic lymphocytes are also susceptible to an RS-dependent lymphocyte-mediated cytotoxic reaction. To identify tumor cell surface antigens that cause the cross-reactive host response, the serum components were analyzed by absorption of RS with allogeneic lymphocytes. RS components were found to recognize allogeneic lymphocyte antigens including Qa-2 and Ly6.2. Specificity for the Qa-2 antigen was further tested using Qa-2-congenic mice. The expression of Qa-2 antigen was detected on the surfaces of MM2 and other tumor cells derived from H-2k mice (seven among nine cell lines tested) by a membrane immunofluorescence method using a Qa-2-specific mAb. Physical characteristics of the Qa-2-specific component in RS were determined and found to differ from those of regular IgGs but to be similar to those of IgDs. Using an enzyme-linked immunosorbent assay with an IgD-specific mAb and Qa-2-lacZ fusion protein, the existence of IgD in RS with specificity for Qa-2 was confirmed. These results suggest that the RS component with Qa-2 specificity is an IgD, the specificity and physiological role of which are unknown.

Allodeterminants and evolution of a novel HLA-B5 CREG antigen, HLA-B SNA.

A novel HLA-B5 CREG gene, HLA-B SNA was cloned and the primary structure was determined. The sequence data showed that HLA-B SNA was identical to HLA-B51 except the alpha 1 domain in which one amino acid substitution at residue 74 and 5 amino acid substitutions associated with the Bw4/Bw6 epitopes were observed between these Ag. The comparison with other HLA-B locus genes suggested that HLA-B SNA evolved from HLA-B51 by gene exchange or recombination at the exon 2 between HLA-B51 and B8. A total of 10 of 14 HLA-B51-specific CTL clones showed significantly weak or no recognition of HLA-B SNA Ag. They also gave the same degree of a lysis of Hmy2CIR cells expressing the HLA-B35/51 chimeric Ag composed of the alpha 1 domain of HLA-B35 and other domains of HLA-B51 as that of Hmy2CIR cells expressing the HLA-B SNA Ag. These results demonstrated that amino acid substitutions within positions 77-83 associated with the HLA-Bw4/Bw6 epitopes have an influence on recognition of the HLA-B SNA antigen by HLA-B51-specific CTL.