MafB promotes atherosclerosis by inhibiting foam-cell apoptosis.

MafB is a transcription factor that induces myelomonocytic differentiation. However, the precise role of MafB in the pathogenic function of macrophages has never been clarified. Here we demonstrate that MafB promotes hyperlipidemic atherosclerosis by suppressing foam-cell apoptosis. Our data show that MafB is predominantly expressed in foam cells found within atherosclerotic lesions, where MafB mediates the oxidized LDL-activated LXR/RXR-induced expression of apoptosis inhibitor of macrophages (AIM). In the absence of MafB, activated LXR/RXR fails to induce the expression of AIM, a protein that is normally responsible for protecting macrophages from apoptosis; thus, Mafb-deficient macrophages are prone to apoptosis. Haematopoietic reconstitution with Mafb-deficient fetal liver cells in recipient LDL receptor-deficient hyperlipidemic mice revealed accelerated foam-cell apoptosis, which subsequently led to the attenuation of the early atherogenic lesion. These findings represent the first evidence that the macrophage-affiliated MafB transcription factor participates in the acceleration of atherogenesis.

Aldehyde oxidase 1 gene is regulated by Nrf2 pathway.

Aldehyde oxidase is a member of the molybd-flavo enzyme family that catalyzes the hydroxylation of heterocycles and the oxidation of aldehydes into corresponding carboxylic acids. Aldehyde oxidase-1 (AOX1) is highly expressed in liver and is involved in the oxidation of a variety of aldehydes and nitrogenous heterocyclic compounds, including anti-cancer and immunosuppressive drugs. However, the physiological substrates of AOX1 have not been identified, and it was unknown how the expression of AOX1 is regulated. Here, we found that the AOX1 gene is regulated by the Nrf2 pathway. Two Nrf2 binding consensus elements (antioxidant responsive element, ARE) are located in the 5' upstream region of the rat AOX1 gene. Molecular analyses using reporter transfection analysis, EMSA, and ChIP analysis show that Nrf2 binds to and strongly activates the rat AOX1 gene.

Frequent downregulation or loss of CD79a expression in plasma cell myelomas: potential clue for diagnosis.

Plasma cell myeloma is a frequent hematogeneous disorder that occurs mainly in older people. Not only bone marrow smears but also clots and/or biopsied specimens are often taken for confirmation of pathological diagnosis. Some specimens show sheet-like plasma cell proliferation associated with immunoglobulin monotype on immunohistology, which readily leads to diagnosis, but many samples do not clearly show light-chain restriction. The aim of the present study was therefore to examine CD79a expression because some samples had reduced expression or none at all. The immunoreactivity of CD79a was categorized into three groups: positive, weakly positive and negative, compared with scattering non-neoplastic plasma cells in the same specimen. Out of 100 specimens of plasma cell myeloma, 48% were positive for CD79a, 15% were weakly positive, and 37% were negative. In contrast, overexpression of cyclinD1 was detected in 26% of examined samples. CD79a-negative cases had a significantly lower percentage of positive staining for cyclinD1 than CD79a-positive or weakly positive cases. Clinicopathological data showed that CD79a-negative expression was associated with decreased platelet numbers in patients. The present study indicates that downregulation or loss of CD79a and/or overexpression of cyclin D1, observed in 59% of neoplastic plasma cell samples, could provide a strong diagnostic clue without regard to the results of immunoglobulin light-chain restriction.

Two novel NKG2D ligands of the mouse H60 family with differential expression patterns and binding affinities to NKG2D.

H60, originally described as a dominant minor histocompatibility Ag, is an MHC class I-like molecule that serves as a ligand for the NKG2D receptor. In the present study, we identified two novel mouse chromosome 10-encoded NKG2D ligands structurally resembling H60. These ligands, which we named H60b and H60c, encode MHC class I-like molecules with two extracellular domains. Whereas H60b has a transmembrane region, H60c is a GPI-anchored protein. Recombinant soluble H60b and H60c proteins bound to NKG2D with affinities typical of cell-cell recognition receptors (K(d) = 310 nM for H60b and K(d) = 8.7 muM for H60c). Furthermore, expression of H60b or H60c rendered Ba/F3 cells susceptible to lysis by NK cells, thereby establishing H60b and H60c as functional ligands for NKG2D. H60b and H60c transcripts were detected only at low levels in tissues of healthy adult mice. Whereas H60b transcripts were detectable in various tissues, H60c transcripts were detected mainly in the skin. Infection of mouse embryonic fibroblasts with murine cytomegalovirus induced expression of H60b, but not H60c or the previously known H60 gene, indicating that transcriptional activation of the three types of H60 genes is differentially regulated. The present study adds two new members to the current list of NKG2D ligands.

c-Maf expression in angioimmunoblastic T-cell lymphoma.

The oncogene c-Maf was recently found to be overexpressed in approximately 50% of multiple myeloma cases, and a role for c-Maf in promoting cyclin D2 expression has been postulated. We previously examined c-Maf expression in various T-cell lymphomas by reverse-transcription polymerase chain reaction and found extremely elevated c-Maf levels in angioimmunoblastic T-cell lymphoma (AILT). In this study, we examined T-cell lymphomas for c-Maf and cyclin expression immunohistochemically. Of 93 cases of T-cell lymphomas we investigated in the current study, c-Maf expression was seen in 23 out of 31 cases of AILT, 3 out of 11 of adult T-cell leukemia/lymphoma, 4 out of 19 of peripheral T-cell lymphoma, unspecified [PTCL(U)], and 0 out of 11 cases of mycosis fungoides, 0 out of 11 of anaplastic large cell lymphoma, and 1 out of 10 of extranodal NK/T-cell lymphoma, nasal type. Double immunostaining in AILT revealed that the majority of c-Maf-positive cells were also positive for CD43 (MT1), CD45RO (UCHL-1), and CD4 but were negative for CD20 (L26). Additionally, cyclins D1 and D2, which stimulate cell cycle progression, were overexpressed in a large number of the c-Maf-positive AILT samples. Quantitative reverse-transcription polymerase chain reaction analysis also showed that c-Maf was overexpressed in 8/31 cases of AILT, 0/19 cases of PTCL(U), 0/11 cases of anaplastic large cell lymphoma, 0/10 cases of extranodal NK/T-cell lymphoma, nasal type, and 2/8 cases of multiple myeloma, presenting significant difference between AILT and PTCL(U) (P=0.016, chi test). These findings strongly suggest that CD4-positive neoplastic T cells in AILT show c-Maf expression and provide new insight into the pathogenesis of AILT suggesting c-Maf to be a useful diagnostic marker for AILT.

Regulation of glutathione transferase P: a tumor marker of hepatocarcinogenesis.

Placental glutathione transferase (GST-P) is specifically expressed during rat haptocarcinogenesis, and has been used as a reliable tumor marker for experimental hepatocarcinogenesis in the rat. The regulation of this tumor marker gene may be associated with the process of carcinogeneisis. By elucidating the mechanisms of such tumor marker gene expression, we may shed light on the molecular mechanisms of carcinogenesis. We analyzed the regulation of the GST-P gene and found that the strong enhancer element GPE1 (GST-P enhancer-1) specifically regulates the GST-P gene by interacting with specific transcription factors in normal liver and during hepatocarcinogenesis. In particular, C/EBPalpha was required for the suppression of GST-P gene in normal liver, whereas the Nrf2/MafK heterodimer was required for the activation of this gene during hepatocarcinogenesis. In this Mini-Review, we describe the positive and negative regulatory mechanisms in the pre-cancerous and normal liver, respectively.

Histone acetyltransferase MOZ acts as a co-activator of Nrf2-MafK and induces tumour marker gene expression during hepatocarcinogenesis.

HATs (histone acetyltransferases) contribute to the regulation of gene expression, and loss or dysregulation of these activities may link to tumorigenesis. Here, we demonstrate that expression levels of HATs, p300 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] were decreased during chemical hepatocarcinogenesis, whereas expression of MOZ (monocytic leukaemia zinc-finger protein; MYST3)--a member of the MYST [MOZ, Ybf2/Sas3, Sas2 and TIP60 (Tat-interacting protein, 60 kDa)] acetyltransferase family--was induced. Although the MOZ gene frequently is rearranged in leukaemia, we were unable to detect MOZ rearrangement in livers with hyperplastic nodules. We examined the effect of MOZ on hepatocarcinogenic-specific gene expression. GSTP (glutathione S-transferase placental form) is a Phase II detoxification enzyme and a well-known tumour marker that is specifically elevated during hepatocarcinogenesis. GSTP gene activation is regulated mainly by the GPE1 (GSTP enhancer 1) enhancer element, which is recognized by the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2)-MafK heterodimer. We found that MOZ enhances GSTP promoter activity through GPE1 and acts as a co-activator of the Nrf2-MafK heterodimer. Further, exogenous MOZ induced GSTP expression in rat hepatoma H4IIE cells. These results suggest that during early hepatocarcinogenesis, aberrantly expressed MOZ may induce GSTP expression through the Nrf2-mediated pathway.

Identification of CCAAT enhancer binding protein alpha binding sites on the human alpha-fetoprotein gene.

Development- and tissue-specific alpha-fetoprotein (AFP) gene expression is controlled by various transcription factors including hepatocyte nuclear factors (HNFs), and a number of cis-acting elements. We recently identified multiple CCAAT/enhancer binding protein (C/EBP) binding sites in the enhancer of the human AFP gene. In this study, we have identified and functionally characterized seven C/EBPalpha-binding sites in the promoter and enhancer regions. An electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis identified two and five C/EBPalpha-binding sites located in the promoter and enhancer regions, respectively. Chromatin immunoprecipitation analyses showed that C/EBPalpha binds both enhancer and promoter regions of the AFP gene in human AFP-producing hepatoma and stomach cancer cells, but not in non-AFP-producing cells. Reporter transfection assays showed that transcription was stimulated by C/EBPalpha binding to each of the elements. These results indicate that C/EBPalpha regulates AFP gene expression through direct binding to multiple sites in the human AFP gene in cultured human cells.

CCAAT enhancer-binding protein alpha suppresses the rat placental glutathione S-transferase gene in normal liver.

The rat placental glutathione S-transferase (GST-P), an isozyme of glutathione S-transferase, is not expressed in normal liver but is highly induced at an early stage of chemical hepatocarcinogenesis and in hepatomas. Recently, we reported that the NF-E2 p45-related factor 2 (Nrf2)/MafK heterodimer binds to GST-P enhancer 1 (GPE1), a strong enhancer of the GST-P gene, and activates this gene in preneoplastic lesions and hepatomas. In addition to the positive regulation during hepatocarcinogenesis, negative regulatory mechanisms might work to repress GST-P in normal liver, but this remains to be clarified. In this work, we identify the CCAAT enhancer-binding protein alpha (C/EBPalpha) as a negative regulator that binds to GPE1 and suppresses GST-P expression in normal liver. C/EBPalpha binds to part of the GPE1 sequence, and the binding of Nrf2/MafK and C/EBPalpha to GPE1 is mutually exclusive. In a transient-transfection analysis, C/EBPalpha activated GPE1 in F9 embryonal carcinoma cells but strongly inhibited GPE1 activity in hepatoma cells. The expression of C/EBPalpha was specifically suppressed in GST-P-positive preneoplastic foci in the livers of carcinogentreated rats. A chromatin immunoprecipitation analysis showed that C/EBPalpha bound to GPE1 in the normal liver in vivo but did not bind in preneoplastic hepatocytes. Introduction of the C/EBPalpha gene fused with the estrogen receptor ligand-binding domain into hepatoma cells, and subsequent activation by beta-estradiol led to the suppression of endogenous GST-P expression. These results indicate that C/EBPalpha is a negative regulator of GST-P gene expression in normal liver.

Activation of connective tissue growth factor gene by the c-Maf and Lc-Maf transcription factors.

The Maf family of transcription factors is expressed during development of various organs and tissues, and is involved in a variety of developmental and cellular differentiation processes. We previously found that c-maf and mafB are strongly expressed in hypertrophic chondrocytes during cartilage development. Connective tissue growth factor (CTGF) is also expressed in hypertrophic chondrocytes. Adenovirus mediated introduction of c-maf gene into the mouse fibroblast cell line C3H10T1/2 strongly induced CTGF expression. CTGF can be induced by TGF-beta via the SMAD pathway; however, the c-Maf could not induce TGF-beta, nor could TGF-beta induce the c-Maf, suggesting that activation of CTGF by Maf is TGF-beta independent. Reporter transfection analysis using C3H10T1/2 cells shows that c-Maf stimulates a CTGF reporter gene. Lc-Maf, a splice variant of c-Maf containing an extra 10 amino acids in the carboxyl terminus, was a stronger inducer of the CTGF reporter gene than c-Maf. Chromatin immunoprecipitation analysis showed that c-Maf binds to the promoter region of the CTGF gene, indicating that Maf directly activates the CTGF gene. Taken together, these data indicate that the CTGF gene is a target of c-Maf and Lc-Maf in cartilage development.

Immunolocalization of cyclin D1 in the developing lens of c-maf -/- mice.

The maf gene encodes a transcription factor protein containing a typical basic/leucine zipper domain structure, a motif for protein dimerization and DNA binding. It has been demonstrated that maf family genes have important roles in embryonic development and cellular differentiation. In this study, localization of cyclin D1, one of the cell cycle-related molecules, was examined immunohistochemically in developing lens cells of c-maf knockout (-/-) mice. At embryonic day 14 in wild-type mice, lens cells consisted of round epithelial cells in a single layer and regularly arranged elongated lens cells, indicating primary lens fiber cells. Cyclin D1-positive nuclei were observed in the lens epithelial cells, whereas cyclin D1 was not detected in the primary lens fiber cells. In c-maf -/- mice, a variety of round epithelial cells were located in the anterior and posterior lens. Many cyclin D1-positive nuclei were observed in lens epithelial cells as well as posterior lens cells. These results are consistent with c-maf playing a role in the regulation of cyclin D1 in developing lens cells.

Mechanisms of a tumor marker, glutathione transferase P, expression during hepatocarcinogenesis of the rat.

The molecular mechanism of any tumor marker expression may shed a light on the mechanism of the particular tumorigenesis. This idea in mind, we have been pursuing the mechanism of specific induction of the placental type glutathione transferase (GST-P) gene during hepatocarcinogenesis of the rat. Making use of advanced technologies of molecular biology including proteomic analysis, gene cloning and production of specific transgenic rats etc., we were able to identify the enhancer and the activator proteins responsible for this tumor marker expression. Negative regulatory regions and modulatory proteins were also found. The overview of this long range study and the future outlook of the problem will be discussed.

Interleukin-6 and mevastatin regulate plasminogen activator inhibitor-1 through CCAAT/enhancer-binding protein-delta.

OBJECTIVE: We sought to determine the etiologic mechanism of proinflammatory cytokine, interleukin-6 (IL-6), and statin as regulators of synthesis of plasminogen activator inhibitor-1 (PAI-1), the physiological fibrinolysis inhibitor and an acute-phase reactant. METHODS AND RESULTS: Transient transfection and luciferase assay in HepG2 human hepatoma-derived cells demonstrated that IL-6 increased PAI-1 promoter activity and mevastatin decreased IL-6-inducible response. Systematic deletion assay of the promoter demonstrated that the region (-239 to -210 bp) containing a putative CCAAT/enhancer-binding protein (C/EBP) binding site was necessary. Point mutation in this site abolished the IL-6-inducible response. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that C/EBPalpha, C/EBPbeta, and C/EBPdelta were involved in protein-DNA complex formation in intact cells. Deoxyribonuclease (DNase) I footprinting analysis revealed that 5' flanking region (-232 to -210 bp) is acute-phase response protein-binding site. C/EBPdelta binding activity was increased by IL-6 and attenuated by mevastatin. Mevastatin attenuated IL-6-mediated increase of C/EBPdelta protein in the nuclear extracts. IL-6 also increased PAI-1 and C/EBPdelta mRNA in mouse primary hepatocytes. CONCLUSIONS: IL-6 increases hepatic PAI-1 expression mediated by the -232- to -210-bp region of the promoter containing a C/EBPdelta binding site. Vascular protection by statins may be partly mediated through regulation of CEBPdelta and consequent modulation of PAI-1 expression.

Regulation of GST-P gene expression during hepatocarcinogenesis.

Placental glutathione S-transferase (GST-P), a member of glutathione S-transferase, is known for its specific expression during rat hepatocarcinogenesis and has been used as a reliable tumor marker for experimental rat hepatocarcinogenesis. To explain the molecular mechanism underlying its specific expression concomitant with the malignant transformation, we have analyzed the regulatory element of the GST-P gene and the transcription factor that binds to this element. From the extensive analyses by the establishment of the transgenic rat lines having various regions of GST-P gene, we could identify the GPE1 as an essential enhancer element for specific GST-P expression. Next, we examined the transcription factor that binds and activates the GPE1, specifically in the early stage of hepatocarcinogenesis and in the hepatoma. Electrophoresis gel mobility shift assay, reporter transfection analysis, and the chromatin immunoprecipitation analysis indicate that the Nrf2/MafK heterodimer binds and activates GPE1 element in preneoplastic lesions and hepatomas but not in the normal liver cells. In this chapter, we describe details of the transgenic rat analyses and the identification of a factor responsible for the specific expression of the GST-P gene and discuss a possible molecular scenario for malignant transformation and tumor marker gene expression.

Expression of maf-B mRNA in the epithelium around the eyelid closure of the mouse eye at embryonic day 18.

Maf encodes a transcription factor protein containing a typical basic leucine zipper domain structure, a motif for protein dimerization and DNA binding. We examined the expression of maf-B mRNA in the epithelium around the eyelid closure. Expression of maf-B mRNA was examined in C57Bl6 mice at the embryonic stages in 12.5 days of gestation (E12.5) and E18 using in situ hybridization with 35S-labeled antisense riboprobes. In embryos studied 12.5 days postconception, a message specific for maf-B was not detected around the developing eyelid. In contrast, maf-B was strongly expressed in the epithelium of the eyelid closure at E18. Expression of maf-B was strongly noted in the suprabasal differentiating cells derived from the basal layer of the conjunctiva and epidermis. In contrast, basal cells in the eyelid closure and in the epidermis, as well as keratinizing cells, did not express maf-B. These data indicate that maf-B mRNA is expressed during development of the eyelid closure.

Developmental contribution of c-maf in the kidney: distribution and developmental study of c-maf mRNA in normal mice kidney and histological study of c-maf knockout mice kidney and liver.

Maf is a family of oncogenes which encodes a nuclear bZip transcription factor protein and has been originally identified from the avian oncogenic retrovirus, AS42. Maf genes have been reported to have critical roles in embryological development and cellular differentiation. In this study, in situ hybridization with (35)S-labeled antisense riboprobes was used to investigate the distribution of c-maf mRNA in balb/c mouse kidneys from 12 (E12) through 17 days (E17) of gestation and then 1 and 4 weeks after birth. Immunocytochemistry of 4-week-old mouse kidney using anti-c-maf antisera was also performed. Kidney and liver sections from c-maf knockout mice at 4 weeks were stained with hematoxylin-eosin, and their histological features were examined. Expression of c-maf mRNA was first detected on E16 in the renal proximal tubules, and it was expressed through 4 weeks after birth. In the c-maf knockout mice at 4 weeks the cytoplasmic volume of the proximal tubule and liver cell was smaller. These findings suggest that expression of the c-maf gene may be involved in the embryological development and/or cell differentiation of kidney and liver cells.

Functional mapping of tissue-specific elements of the human alpha-fetoprotein gene enhancer.

Serum alpha-fetoprotein (AFP) levels in hepatocellular carcinoma (HCC) patients and expression of the protein in cultured HCC cell lines are highly variable. These observations may arise from features correlated with tissue-specific expression of the gene. Extremely strong and potent liver-specific enhancer activity is confined from -4.1 to -3.3 kb upstream to the human AFP gene in contrast with that of the rodent which exists in three widely separated regions. To understand the tissue-specific expression of AFP, we examined cis-acting elements in the enhancer. Results revealed binding sites for selected liver-enriched transcription factors (LETFs) in both domains A (-4120 to -3756 bp) and B (-3492 to -3300 bp) of the gene. These sites included: one hepatocyte nuclear factor (HNF)-1 and HNF-4, two HNF-3, and two C/EBP binding sites in domain A. An adjacent domain B contained one HNF-3 site and three C/EBP sites plus a previously identified HNF-1 site. Each of these elements alone has the ability to stimulate heterogeneous promoter activity in a dose-dependent manner when transfected into AFP producing cells. A comparative study showed that the presence of two HNF-1 and one HNF-4 site is a characteristic feature of human but not rodent AFP enhancer. The mRNA levels of the liver-enriched transcription factors (LETFs) were variable in individual HCC cell lines and together with silencer activities may underlie differential expression of the AFP gene.

Expression of c-maf and mafB genes in the skin during rat embryonic development.

The maf oncogene (v-maf) was initially identified in an avian oncogenic retrovirus, AS42, which induces musculoaponeurotic fibrosarcoma in vivo and transforms chicken embryo fibroblasts in vitro. Genes of the maf family have important roles in embryonic development and cellular differentiation. Both genes are expressed in a wide variety of tissues including spleen, kidney, lens and liver. The present study was performed to analyze expression of c-maf-1 and mafB genes in skin of embryonic stages from 15 days onwards using in situ hybridization. Expression of c-maf mRNA was first detected on embryonic day (ED) 16 in the nuclei of cells in the basal layer in developing epidermis. On ED 19, high expression was detected in the nucleus of basal keratinocytes and developing hair germs. On postnatal day (PD) 3, expression of c-maf had disappeared in epidermis and hair follicles. MafB showed similar expression patterns as c-maf. Our findings indicate that c-maf and mafB are involved in embryonic development of epidermis and hair follicles.

Transcription factor Nrf2/MafK regulates rat placental glutathione S-transferase gene during hepatocarcinogenesis.

The rat GST-P (placental glutathione S-transferase), a phase II detoxifying enzyme, is not expressed in normal liver cells, but is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells. Results of previous studies indicated that GST-P gene activation was mainly controlled by an enhancer element, GPE1 (GST-P enhancer 1), but the specific activation mechanism of the GST-P gene was not fully understood [Morimura, Suzuki, Hochi, Yuki, Nomura, Kitagawa, Nagatsu, Imagawa and Muramatsu (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2065-2068; Suzuki, Imagawa, Hirabayashi, Yuki, Hisatake, Nomura, Kitagawa and Muramatsu (1995) Cancer Res. 55, 2651-2655]. In the present study, we investigate the transcription factor Nrf2/MafK heterodimer (where Nrf2 stands for NF-E2 p45-related factor 2) as an activator of the GST-P gene through the action of GPE1 during hepatocarcinogenesis. Electrophoretic mobility-shift assay and footprinting analysis with wild-type GPE1 and GPE1 point mutants showed that the Nrf2/MafK heterodimer specifically bound GPE1. Reporter transfection assays indicated that Nrf2 strongly stimulated GST-P gene expression in mouse F9 embryonal carcinoma cells and H4IIE rat hepatoma cells. Northern-blot analysis indicated that GST-P and Nrf2 mRNA increased in parallel with development of precancerous lesions and hepatocellular carcinoma. Keap1 (Kelch-like ECH-associated protein 1), an inhibitory factor of Nrf2, decreased the activation of GPE1 by Nrf2 and this suppression was restored after treatment with electrophilic compounds. GST-P mRNA expression in H4IIE cells was induced by electrophilic compounds, as was the expression of mRNAs of other phase II detoxifying enzymes. Chromatin immunoprecipitation analyses showed that antibodies both against Nrf2 and against MafK precipitated GPE1 from the chromatin of the pre-neoplastic hepatocytes and rat hepatoma cells (H4IIE and dRLh84), but not from normal hepatocytes. These results indicate that the Nrf2/MafK heterodimer regulates GST-P gene expression during early hepatocarcinogenesis and in hepatoma cells.

[Proliferative regulation in the cornea and lens].

PURPOSE: To examine the mechanism of regulation of proliferation in epithelial scraped cornea and the developing lens. METHOD AND RESULTS: C57B16 mouse, p27 (KIP 1)-/- mice, Skp2-/- mice and Skp2-/-/p27 (KIP 1)-/- double knockout mice were examined by immunocytochemistry using anti-p27 (KIP1) antibody, and cells in the "S" phase of DNA synthesis were analyzed by immunocytochemistry using anti-BrdU antibody. The p 27 (KIP 1) was expressed in basal cells of the central and peripheral region of the cornea and limbus. This expression was not detected 24 hr after epithelial scraping, when there were many cells in the "S" phase of DNA synthesis in the corneal epithelium. There was no obvious difference in the thickness and anti-BrdU staining in the corneal epithelium of p 27(KIP 1)-/- mice from that of controls. 24 hr after the epithelial scraping in the Skp 2-/- mice, the corneal epithelium was thinner than in wild-type mice and had many p 27(KIP 1) positive cells and few BrdU positive cells. In contrast, 24 hr after the epithelial scraping in the Skp 2-/-/p 27(KIP 1)-/- double knockout mice, the corneal epithelium was as thick as in wild-type mice and had many BrdU positive cells. CONCLUSIONS: These results suggest that degradation of p27(KIP1) by Skp 2 is involved in the regulation of proliferation in response to wounding of the corneal epithelium. To examine the involvement of the c-maf gene in the proliferation of the lens cells, eyes of the E13 and E18 stages of wild-type and c-maf-/- mice were analyzed by BrdU incorporation assay, TUNEL assay, and immunocytochemistry using an anti-P 27 (KIP 1) and an anti-P 57 (KIP 2) antibody. In the E 13 and E 18 c-maf mutant lens, BrdU-positive cells were detected at the posterior region of the lens. Cell-cycle inhibitor P 27 (KIP 1) and P 57 (KIP 2) were expressed in the equatorial and posterior region of the lens of both wild-type and c-maf-/- lenses. These results suggest that the expression of c-maf is required for differentiation and cell cycle arrest of lens cells independent of p 27 (KIP 1) and p 57 (KIP 2).