Blockage of Akt activation suppresses cadmium-induced renal tubular cellular damages through aggrephagy in HK-2 cells.

We have reported that an environmental pollutant, cadmium, promotes cell death in the human renal tubular cells (RTCs) through hyperactivation of a serine/threonine kinase Akt. However, the molecular mechanisms downstream of Akt in this process have not been elucidated. Cadmium has a potential to accumulate misfolded proteins, and proteotoxicity is involved in cadmium toxicity. To clear the roles of Akt in cadmium exposure-induced RTCs death, we investigated the possibility that Akt could regulate proteotoxicity through autophagy in cadmium chloride (CdCl2)-exposed HK-2 human renal proximal tubular cells. CdCl2 exposure promoted the accumulation of misfolded or damaged proteins, the formation of aggresomes (pericentriolar cytoplasmic inclusions), and aggrephagy (selective autophagy to degrade aggresome). Pharmacological inhibition of Akt using MK2206 or Akti-1/2 enhanced aggrephagy by promoting dephosphorylation and nuclear translocation of transcription factor EB (TFEB)/transcription factor E3 (TFE3), lysosomal transcription factors. TFEB or TFE3 knockdown by siRNAs attenuated the protective effects of MK2206 against cadmium toxicity. These results suggested that aberrant activation of Akt attenuates aggrephagy via TFEB or TFE3 to facilitate CdCl2-induced cell death. Furthermore, these roles of Akt/TFEB/TFE3 were conserved in CdCl2-exposed primary human RTCs. The present study shows the molecular mechanisms underlying Akt activation that promotes cadmium-induced RTCs death.

The incidence and mechanism of sunitinib-induced thyroid atrophy in patients with metastatic renal cell carcinoma.

BACKGROUND: To elucidate the incidence and mechanisms of sunitinib-induced thyroid atrophy, we investigated serial volumetric and functional changes, and evaluated histological changes of the thyroid gland in metastatic renal cell carcinoma patients who received sunitinib. METHODS: Thyroid volume (by computed tomography volumetry) and thyroid function were measured at baseline, during the treatment, and at post-treatment periods. Histological evaluation of the thyroid gland was performed in four autopsied patients. RESULTS: The median reduction rate in thyroid volume at last evaluation during sunitinib treatment was 30% in all 17 patients. The incidence of hypothyroidism during sunitinib treatment was significantly higher in the high reduction rate group (n=8; more than 50% reduction in volume) than in the low reduction rate group (n=9; less than 50% reduction in volume). Half of the patients in the high reduction rate group exhibited a transient thyroid-stimulating hormone suppression, suggesting thyrotoxicosis during sunitinib treatment. Histological evaluation demonstrated atrophy of thyroid follicles and degeneration of follicular epithelial cells without critical diminution of vascular volume in the thyroid gland. CONCLUSION: Thyroid atrophy is frequently observed following sunitinib treatment and may be brought about by sunitinib-induced thyrotoxicosis or the direct effects of sunitinib that lead to degeneration of thyroid follicular cells.

Short communication: expression of human endogenous retrovirus-R gene links to differentiation of squamous cells.

To investigate the biological roles of human endogenous retrovirus-R (HERV-R) in vivo, we established transgenic rats carrying the full sequence of the viral genome with control of its own long terminal repeat promoter. The Env protein was expressed on the surface of the epidermis of fetal HERV-R transgenic rats on day 10 of gestation. The epidermal Env expression disappeared by day 18 of gestation. After day 18 of gestation, the Env protein was detected in the prickle layer of the esophageal epithelium of transgenic rats. Interestingly, it was not detected in the basal layer of the epithelium, and the expression in the granular layer was weaker than in the prickle layer. These findings suggest that expression of HERV-R is linked not only to the development but also to the differentiation of squamous cells. Next, we examined alterations in the expression of the HERV-R env gene in cultured human squamous cells after exposure to all-trans retinoic acids (ATRA). The env expression was increased by ATRA in a dose-dependent manner, while the expression of transglutaminase 1 (TGM1), a terminal marker for squamous differentiation, was decreased. TGM1 is expressed in the granular layer of the squamous epithelium, and ATRA suppresses the differentiation of cultured squamous cells. Thus, these in vitro data also suggest that HERV-R expression is regulated by a mechanism closely related to the differentiation of squamous cells. This study is the first to demonstrate the association of HERV-R expression and differentiation of squamous cells.

Articular tissues expressing the env-pX transgene are required for generation of arthritogenic T cells in human T cell leukemia virus type I transgenic rats.

OBJECTIVE: Human T cell leukemia virus type I env-pX transgenic rats (env-pX rats) were used to investigate the pathogenesis of arthritis. METHODS: Phenotype of cells infiltrated into arthritic joints in env-pX rats was analyzed using flow cytometry and cell-transfer experiments were done using env-pX and wild-type WKAH rats. RESULTS: The majority of T cells infiltrated into arthritic joints in env-pX rats exhibited a CD4 and activated phenotype. Transfer of these T cells into articular space in wild-type WKAH rats succeeded to induce arthritis similarly seen in env-pX rats. However, injection of the cells into sites other than joints did not induce inflammation. Transfer of in vitro-stimulated lymph node cells from disease-free env-pX rats into articular space did not induce arthritis in wild-type WKAH rats. CONCLUSION: These findings suggest that articular tissues carrying the env-pX transgene are required for generation of arthritogenic T cells in env-pX rats. However, the constitutive antigens other than the transgene products are recognized as immunological targets by the arthritogenic T cells in the advanced arthritic joints. Molecules expressed specifically in articular tissues may be needed to maintain the inflammatory cell infiltration.

Elution of IgA from kidney tissues exhibiting glomerular IgA deposition and analysis of antibody specificity.

Glomerular IgA deposits were eluted from renal biopsy specimens exhibiting IgA nephropathy (IgAN) by using a combination of citrate buffer and collagenase. Collagenase predigestion of the kidney tissues resulted in increased amounts of IgA eluted by citrate buffer, and the elusion procedure did not attenuate the antigen-binding ability of IgA antibody. When reactivity of the eluted IgA with bacteria components was examined by Western blotting, the most notable reaction was observed for Haemophilus influenzae lysates in the form of a 34 kD-band. The reactivity of IgA eluted from the kidney tissues against the H. influenzae 34 kD antigen was evident in 3 of 5 IgAN cases. However, similar reactivity was also evident in 2 of 6 non-IgAN hepatic diseases exhibiting a glomerular IgA deposition. These findings suggest that antibody specificity of IgA against H. influenzae itself may not be directly associated with glomerular injury, although anti-H. influenzae 34 kD IgA was deposited in the kidney, at least in part, by IgAN. Further investigations into the properties of IgA deposited in the glomerulus are needed. Our improved method for IgA elution from kidney tissues would be useful for analysing the pathogenesis of IgAN.

Immunological hyperresponsiveness in HTLV-I LTR-env-pX transgenic rats: a prototype animal model for collagen vascular and HTLV-I-related inflammatory diseases.

We have earlier reported that diverse collagen vascular diseases, including arthritis, arteritis, thrombosis, myocarditis, myositis, sialo-/dacryoadenitis and dermatitis develop with the advent of autoantibodies in transgenic rats carrying the LTR-env-pX gene of human T lymphocyte virus type I (LTR-env-pX rats). To clarify the pathogenesis of these collagen vascular diseases, immunological features of LTR-env-pX rats were examined. In LTR-env-pX rats affected with these diseases, expression of CD80/86 on both tissue-infiltrating and peripheral T cells increased, compared with findings in non-transgenic rats with experimental inflammatory diseases. CD80/86 was also upregulated on peripheral T cells in LTR-env-pX rats prior to the development of diseases. Lymphocytes from LTR-env-pX rats showed an increase in autologous proliferation and were hyperreactive against several mitogens, including concanavalin A, immobilized anti-CD3 antibodies, and superantigens in vitro. Antigen-specific immune response was also enhanced in LTR-env-pX rats. The collective evidence indicates that lymphocytes of LTR-env-pX rats constitutively express surface molecules related to T cell activation and are immunologically hyperresponsive. Bone marrow cell transfer from LTR-env-pX rats to lethally irradiated non-transgenic rats revealed that these immunologically pre-activated and hyperresponsive lymphocytes play a critical role in the pathogenesis of several collagen vascular diseases, especially of dermatitis in LTR-env-pX rats.

Anti-Thy-1 monoclonal antibody with specific reactivity with vascular endothelial cells in rat glomeruli.

Inoculation with anti-Thy-1 antibodies (Abs) in rats induces glomerulonephritis resembling human mesangiolytic and/or mesangioproliferative diseases. Some anti-Thy-1 monoclonal Abs (mAbs) react with both mesangial and glomerular endothelial cells, whereas others react solely with mesangial cells in rat kidney. These findings suggest that the rat Thy-1 molecule possesses at least 2 variant forms, including a mesangial and a vascular endothelial isoform. However, anti-Thy-1 mAbs with specific reactivity with glomerular endothelial cells have not been available. We describe here a unique anti-rat Thy-1 mAb, TM78-8. The epitope for TM78-8 is closely related, but not identical, to that for OX-7, a commercially available anti-rat Thy-1 mAb. Immunoblotting, immunohistochemistry and immunoelectron microscopy confirm that TM78-8 reacts exclusively with Thy-1 antigens on the surface of vascular endothelial cells in rat glomeruli. TM78-8 may be a suitable marker for rat glomerular endothelial cells as well as for the vascular endothelial isoform of the rat Thy-1 molecule. Intravenous injection of TM78-8 did not induce glomerulonephritis in rats, whereas OX-7 did, indicating that TM78-8 is not nephritogenic. This finding also corresponds with the current consensus that Thy-1 antigens expressed on mesangial cells play an essential role in the development of Thy-1 nephritis.

Transduction of a murine dominant negative activation transcription factor 1 increases cell surface expression of the class I MHC on a human epidermoid tumor cell line.

The transcription of the MHC class I genes is regulated by interaction of cis-elements, located in the 5' genomic flanking regions, with sequence-specific trans-factors. We have identified a cis-regulatory element, 5'-TGACGCG-3', of the H-2D(d) gene. This cyclic adenosine-3',5'-monophosphate regulatory element (CRE)-like sequence, named H-2 binding factor 1 (H-2 BF1) binding motif, is highly conserved among species. In addition, we found that homo- and heterodimers of activation transcription factor 1 (ATF-1) and CRE binding protein (CREB) associate with the H-2 BF1 binding motif and activate transcription of the H-2D(d) gene. Here we demonstrate that a homologue of ATF-1, originally isolated and designated ATF-1DN, acts as a dominant repressor, blocking the ability of wild-type ATF-1 and CREB to bind to the H-2 BF1 probe in electrophoretic mobility shift assays (EMSA). We have utilized this molecule to analyze the participation of the H-2 BF1 complexes, consisting of the H-2 BF1 binding motif and ATF-1/CREB trans-factors, in the physiological regulation of MHC class I expression in tissue culture cells. A human epidermoid carcinoma cell line, A431, was transfected with ATF-1DN and clones expressing the gene transcripts were selected. When analyzed in the EMSA, nuclear proteins prepared from these clones exhibited a decreased shift of the H-2 BF1 probe corresponding to the levels of the ATF-1DN gene expression. Additionally, MHC class I expression of cells with reduced H-2 BF1 activity was significantly higher than in control cells lacking ATF-1DN. These findings indicate that in these carcinoma cells, the H-2 BF1 complexes negatively regulate the constitutive expression of MHC class I.

Morphological aspects of ectopia cordis: four case reports and a review of the literature.

Ectopia cordis is a rare congenital anomaly. We present 4 cases of ectopia cordis, 1 of which is the first report of an affected fetus in a triplet pregnancy. The morphological relationship between the types of ectopia cordis and their outcomes were investigated in all 4 cases. In addition, the literature on ectopia cordis in Japan was reviewed and discussed.

Cytokine regulation of env gene expression of human endogenous retrovirus-R in human vascular endothelial cells.

To determine whether human endogenous retroviruses are implicated in the pathogenesis of inflammatory vascular diseases of unknown etiology, we examined mRNA expression of a human endogenous retrovirus, HERV-R, which has a long open reading frame in the env region, in cultured human vascular endothelial and smooth muscle cells stimulated in the presence of various cytokines. mRNA of HERV-R was always evident in these cells but not in fibroblastic cells. Levels of expression in vascular endothelial cells were significantly regulated by treatment with tumor necrosis factor-alpha, interleukin (IL)-1alpha, and IL-1beta as up-regulators and interferon-gamma as a down-regulator. These observations are interpreted to mean that HERV-R expression may be up- or down-regulated at sites of inflammation in vessels in vivo and hence may play a pathogenetic role in inflammatory vascular diseases in humans, perhaps similar to endogenous retroviruses in mouse models of polyarteritis nodosa in humans.

A rare case of acromegaly associated with pachydermoperiostosis.

Pachydermoperiostosis (PDP) is a rare syndrome manifested clinically by finger clubbing, extremity enlargement, hypertrophic skin changes, and periosteal bone formation. The pathogenesis of the disorder has not been clarified and few endocrine abnormalities were apparent. We report here a 58-year-old man with acromegaly associated with PDP, the features of clubbed fingers, coarse skin, and cutis verticis gyrata. Acromegaly due to GH-producing pituitary adenoma was confirmed in endocrinological and pathological studies.

Reducing cytotoxicity induced by Sindbis viral vectors.

Sindbis virus has been recognized as a potentially useful virus vector for gene therapy. In an effort to improve its utility and provide cell-targeting capability to gene therapy vectors, we recently developed Sindbis virus vectors possessing chimeric envelopes with cell-specific targeting ability [K. Ohno et al. Nature Biotechnol 15:763-767, 1997; K. Sawai et al. Biochem Biophys Res Commun 248:315-323, 1998]. However, a residual problem associated with Sindbis virus vectors is the apoptotic effect of this virus on infected cells. To address this issue, we have studied the possible role of bcl-2 expression. Bcl-2 expression has been postulated to facilitate the establishment of persistent Sindbis viral infection by blocking virus-induced apoptosis. In this study we produced a Sindbis virus vector capable of expressing human bcl-2 and the reporter gene, lacZ. This chimeric virus (SinRep/lacZ/bcl-2/DH-BB) showed a marked reduction in induced apoptosis in infected cells. For example, after infection with this vector, cell proliferation of BHK cells was 55% of that of uninfected cells 2 days after infection and 40% 3 days after infection. While this reflected a significant degree of apoptosis, the effect was much less pronounced than that seen with wild-type Sindbis virus. Cell proliferation was reduced to 26% 2 days after wild-type virus infection of BHK cells and to only 7% 3 days after infection. Although additional work will be required to eliminate apoptosis induced by Sindbis virus vectors, the studies reported here suggest that such a goal may be achievable after additional modification of the vectors.

Giant cell granulamatous hypophysitis with remarkable uptake on Gallium-67 scintigraphy.

We treated a man with giant cell granulomatous hypophysitis with pituitary enlargement, as seen on magnetic resonance imaging. Endocrinological examination revealed panhypopituitarism and diabetes insipidus. Microscopic examination of the specimen obtained by transsphenoidal pituitary biopsy revealed a granulomatous lesion, composed of epitheliod cells, Langhans' multinucleated giant cells, lymphocytes and other chronic inflammatory cells. On whole body gallium-67 scintigraphy, there was extensive uptake in the pituitary gland. Gallium-67 scintigraphy may greatly aid in the diagnosis of granulomatous hypophysitis.

The regulation of murine H-2Dd expression by activation transcription factor 1 and cAMP response element binding protein.

Resistance to radiation leukemia virus (RadLV)-induced leukemia is correlated with an increase in H-2Dd expression on the thymocyte surface. It has been shown that elevated H-2Dd expression on infected thymocytes is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5'-flanking sequence (5'-TGACGCG-3') of the H-2Dd gene. Recently, it has been shown that the activation transcription factor 1 (ATF-1) homodimer is one form of the H-2 BF1 complex. Here we demonstrate that the cAMP response element binding protein (CREB) homodimer and the heterodimer of CREB/ATF-1 also recognize the cis regulatory motif and are two additional forms of the H-2 BF1 complex. The levels of mRNA encoding ATF-1 and CREB were both increased in RadLV-infected thymocytes that showed increased levels of H-2 mRNA. Also, all three H-2 BF1 binding activities, ATF-1 homodimer, CREB homodimer, and ATF-1/CREB heterodimer, were increased in RadLV-infected thymocytes that expressed high levels of H-2Dd Ag on the cell surface. Transfection experiments demonstrated that ATF-1 and CREB activated a reporter plasmid containing the H-2 BF1 motif. These observations strongly suggest that both ATF-1 and CREB are involved in the regulation of H-2 gene expression following RadLV infection of mouse thymocytes.

Promotion of early osteoclastogenesis and B lymphopoiesis in the bone marrow of transgenic rats with the env-pX gene of human T-cell lymphotropic virus type I.

Human T-cell lymphotropic virus type I (HTLV-I) is associated with various clinical disorders including adult T cell leukemia, myelopathy, arthropathy. Hypercalcemia resulting from osteoclast activation and a variety of hematopoietic abnormalities have been also observed in HTLV-I infected patients, however, precise mechanism about initial trigger(s) prior to presenting symptoms is still unknown. In this study, to assess effects of HTLV-I on hematopoiesis, we analysed characteristics of early hematopoietic precursors in HTLV-I env-pX transgenic rats. Progenitor cells for osteoclasts were significantly increased even in the marrow of asymptomatic env-pX rats. Progenitors for B cells were also highly enriched, while colony forming cells (CFC) elicited by GM-CSF(CFU-GM) and M-CSF(CFU-M) were comparable to normal littermates. Following arthritis in env-pX transgenic rats, osteoclastogenesis was further augmented and the CFCs were increased. Bone marrow cells carrying adjuvant-induced arthritis retained a constant number of progenitors for osteoclast and B lymphocytes, whereas the number of CFU-GM and CFU-M increased. These results indicate that the env-pX transgene affect early stages of osteoclast and B-cell lineages prior to developing diseases, in contrast, an increase of the CFCs was caused indirectly by arthritis. This study provides a novel standpoint for the mechanisms of pathogenesis by HTLV-I.

Models of HTLV-I-induced diseases. Infectious transmission of HTLV-I in inbred rats and HTVL-I env-pX transgenic rats.

To examine the pathogenic roles of HTLV-I in HTLV-I-induced diseases, we developed two models; namely HTLV-I carrier rats and HTLV-I env-pX transgenic rats. Among life long HTLV-I carriers in seven rat strains, only WKAH rats with the RT1k haplotype developed chronic progressive myeloneuropathy, resembling HAM/TSP clinically and histologically in humans, designated as HAM rat disease and after long incubation periods. Apoptosis of myelin forming cells, oligodendrocytes and Schwann cells associated with HTLV-I infection appears to be the primary cause of HAM rat disease. Local activation of the pX gene and TNF alpha gene was evident in these rats. WKAH rats transgenic for HTLV-I env-pX gene were established and at age 5 weeks, swelling of the bilateral ankle joints began to develop and histological features of the affected joints resembled findings in cases of rheumatoid arthritis (RA): high-titers of rheumatoid factors were present in these rats. A series of vascular collagen diseases such as polyarteritis nodosa-like angiitis, polymyositis, myocarditis, and Sjögren's syndrome-like sialodenitis together with RA were present, even in one individual animal. These transgenic rats as well as HAM rats appear to be suitable animal models for elucidating pathogenic mechanisms implicated in HTLV-I-induced diseases and also various demyelinating vascular collagen diseases of unknown etiology.

A wide spectrum of collagen vascular and autoimmune diseases in transgenic rats carrying the env-pX gene of human T lymphocyte virus type I.

To investigate the pathogenesis of human T lymphocyte virus type I (HTLV-I)- related diseases, the env-pX gene of HTLV-I was introduced into the germline of inbred Wistar-King-Aptekman-Hokudai rats. A wide spectrum of collagen vascular diseases was evident in the transgenic rats, including chronic destructive arthritis similar to rheumatoid arthritis, necrotizing arteritis mimicking polyarteritis nodosa, polymyositis, myocarditis, dermatitis, and chronic sialoadenitis and dacryoadenitis resembling Sjögren's syndrome in humans. Thymic atrophy with the depletion of CD4 and CD8 double-positive thymocytes was also observed. In these animals, a number of autoantibodies, including high titers of rheumatoid factor, were present in the serum. We propose that the HTLV-I env-pX gene region may play a pathogenetic role in the development of collagen vascular and autoimmune diseases associated with autoimmune phenomenon.

Interleukin-1alpha regulates Thy-1 expression on rat vascular endothelial cells.

We recently reported that Thy-1, a surface molecule induced on the rat endothelium, regulates vascular permeability at sites of inflammation. Although the rat inferior vena cava (IVC) did not express Thy-1 in vivo, cultured endothelial cells from the IVC did express Thy-1, thereby suggesting that the expression was acquired during cultivation of the cells in vitro, possibly by autoactivation by cytokine-like substances. Interleukin (IL)-1alpha but not tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma was detected in culture supernatants of rat endothelial cells (REC) by ELISA. The production of IL-1alpha by REC was augmented by exogenously added IL-1alpha, thereby implying the presence of autocrine regulation by IL-1alpha. The unaltered expression of Thy-1 by exogenously added IL-1alpha suggests that Thy-1 expression on REC had already been maximally induced by autologous cytokines; the expression of Thy-1 on REC was lowered by inhibiting protein kinase C and by depleting IL-1alpha activity from culture supernatants. Although cytokine-like regulators, other than IL-1alpha, TNF-alpha, or IFN-gamma, produced by REC may also modulate the expression of Thy-1, it is at least in part mediated by IL-1alpha in vitro. Moreover, Thy-1 expression was induced on rat vascular endothelium at the subcutis where recombinant IL-1alpha was injected. The evidence indicates that IL-1alpha functions as one regulator responsible for the induction of Thy-1 on REC, in vitro as well as in vivo.