Nobutaka Ohgami

Ret-Dependent Cell Rearrangements in the Wolffian Duct Epithelium Initiate Ureteric Bud Morphogenesis

While the genetic control of renal branching morphogenesis has been extensively described, the cellular basis of this process remains obscure. GDNF/RET signaling is required for ureter and kidney development, and cells lacking Ret are excluded from the tips of the branching ureteric bud in chimeric kidneys. Here, we find that this exclusion results from earlier Ret-dependent cell rearrangements in the caudal Wolffian duct, which generate a specialized epithelial domain that later emerges as the tip of the primary ureteric bud. By juxtaposing cells with elevated or reduced RET activity, we find that Wolffian duct cells compete, based on RET signaling levels, to contribute to this domain. At the same time, the caudal Wolffian duct transiently converts from a simple to a pseudostratified epithelium, a process that does not require Ret. Thus, both Ret-dependent cell movements and Ret-independent changes in the Wolffian duct epithelium contribute to ureteric bud formation.

Niemann-Pick Type C Disease and Intracellular Cholesterol Trafficking

Ta-Yuan Chang‡§, Patrick C. Reid¶, Shigeki Sugii , Nobutaka Ohgami‡, Jonathan C. Cruz**, and Catherine C. Y. Chang‡ From the ‡Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, ¶Department of Molecular Biology and Medicine, RCAST, University of Tokyo, Tokyo 153-8904, Japan, Gene Expression Laboratory, Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, California 92037, and **Department of Pathology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115

CD36, serves as a receptor for advanced glycation endproducts (AGE).

Interaction of advanced glycation endproducts (AGE) with AGE receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE receptors identified so far are receptor for AGE (RAGE), 80 K-H, OST-48, galectin-3, and macrophage scavenger receptor, types I and II (SR-A) [Eur. J. Biochem. 230 (1995) 408; Nature 386 (1997) 292.]. Since SR-A is known to belong to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to class B scavenger receptor family (SR-B) can recognize AGE proteins as a ligand. This was tested in the present study at the cellular level by using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CHO-CD36 cells). 125I-AGE-bovine serum albumin (BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-CD36, but not wild-type CHO cells. Endocytic uptake of 125I-AGE-BSA by these cells was inhibited 50% by oxidized low-density lipoprotein (Ox-LDL) and 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of Ox-LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major Ox-LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox-LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

CD36-mediated endocytic uptake of advanced glycation end products (AGE) in mouse 3T3-L1 and human subcutaneous adipocytes

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. We here show that AGE‐modified bovine serum albumin (BSA) is endocytosed by adipocytes via CD36. Upon differentiation, 3T3‐L1 and human subcutaneous adipose cells showed marked increases in endocytic uptake and subsequent degradation of [125I]AGE‐BSA, which were inhibited effectively by the anti‐CD36 antibody. Ligand specificity of CD36 for modified BSAs was compared with that of LOX‐1 and scavenger receptor class A. Effect of fucoidan on [125I]AGE‐BSA binding showed a sharp contrast to that on [125I]‐oxidized low density lipoprotein. These results implicate that CD36‐mediated interaction of AGE‐modified proteins with adipocytes might play a pathological role in obesity or insulin‐resistance.

RAS/RAF/MEK/ERK and PI3K/PTEN/AKT Signaling in Malignant Melanoma Progression and Therapy

Cutaneous malignant melanoma is one of the most serious skin cancers and is highly invasive and markedly resistant to conventional therapy. Melanomagenesis is initially triggered by environmental agents including ultraviolet (UV), which induces genetic/epigenetic alterations in the chromosomes of melanocytes. In human melanomas, the RAS/RAF/MEK/ERK (MAPK) and the PI3K/PTEN/AKT (AKT) signaling pathways are two major signaling pathways and are constitutively activated through genetic alterations. Mutations of RAF, RAS, and PTEN contribute to antiapoptosis, abnormal proliferation, angiogenesis, and invasion for melanoma development and progression. To find better approaches to therapies for patients, understanding these MAPK and AKT signaling mechanisms of melanoma development and progression is important. Here, we review MAPK and AKT signaling networks associated with melanoma development and progression.

CD36, a Member of Class B Scavenger Receptor Family, Is a Receptor for Advanced Glycation End Products

Abstract: Interaction of advanced glycation end products (AGE) with AGE‐receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE‐receptors identified so far are RAGE (receptor for AGE), 80 K‐H, OST‐48, galectin‐3, and SR‐A (macrophage scavenger receptor type I and II). Since SR‐A belongs to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to the class B scavenger receptor family (SR‐B) can recognize AGE‐proteins as a ligand. This was tested in the present study at the cellular level using CHO (Chinese hamster ovary) cells overexpressing human CD36 (CHO‐CD36 cells). 125I‐AGE‐BSA (bovine serum albumin) was endocytosed in a dose‐dependent fashion and underwent lysosomal degradation by CHO‐CD36 but not wild‐type CHO cells. Endocytic uptake of 125I‐AGE‐BSA by these cells was inhibited 50% by oxidized LDL (Ox‐LDL) and 60% by FA6‐152, an anti‐CD36 antibody inhibiting cellular binding of Ox‐LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE‐proteins. Because CD36 is one of the major Ox‐LDL receptors and is upregulated in macrophage‐ and smooth muscle cell‐derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox‐LDL, AGE‐proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

Molecular Network Associated with MITF in Skin Melanoma Development and Progression

Various environmental and genetic factors affect the development and progression of skin cancers including melanoma. Melanoma development is initially triggered by environmental factors including ultraviolet (UV) light, and then genetic/epigenetic alterations occur in skin melanocytes. These first triggers alter the conditions of numerous genes and proteins, and they induce and/or reduce gene expression and activate and/or repress protein stability and activity, resulting in melanoma progression. Microphthalmia-associated transcription factor (MITF) is a master regulator gene of melanocyte development and differentiation and is also associated with melanoma development and progression. To find better approaches to molecular-based therapies for patients, understanding MITF function in skin melanoma development and progression is important. Here, we review the molecular networks associated with MITF in skin melanoma development and progression.

Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (θ-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCθ can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCθ signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCθ. Under this condition, BCθ mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCθ staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to β-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCθ staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCθ is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.

c-Ret–mediated hearing loss in mice with Hirschsprung disease

A significantly increased risk for dominant sensorineural deafness in patients who have Hirschsprung disease (HSCR) caused by endothelin receptor type B and SOX10 has been reported. Despite the fact that c-RET is the most frequent causal gene of HSCR, it has not been determined whether impairments of c-Ret and c-RET cause congenital deafness in mice and humans. Here, we show that impaired phosphorylation of c-Ret at tyrosine 1062 causes HSCR-linked syndromic congenital deafness in c-Ret knockin (KI) mice. The deafness involves neurodegeneration of spiral ganglion neurons (SGNs) with not only impaired phosphorylation of Akt and NF-κB but decreased expression of calbindin D28k in inner ears. The congenital deafness involving neurodegeneration of SGNs in c-Ret KI mice was rescued by introducing constitutively activated RET. Taken together with our results for three patients with congenital deafness with c-RET–mediated severe HSCR, our results indicate that c-Ret and c-RET are a deafness-related molecule in mice and humans.

Effects of light smoking on extra-high-frequency auditory thresholds in young adults.

There have been few reports showing a correlation between hearing levels and life style in young people. In this study, we succeeded in sensitively evaluating hearing levels in 51 young male adults of 21-23 years in age by 12 k Hz extra-high-frequency auditory thresholds, which cannot be measured by usual audiometry devices for clinical use. Noise exposure, alcohol consumption and sleeping time did not affect hearing levels in young adults. Auditory thresholds of 12 kHz frequency in smokers were significantly (p < 0.05) higher than those in non-smokers, while there were no differences in 1 kHz, 4 kHz and 8 kHz frequencies of hearing levels between smokers and non-smokers. Since the Brinkman Index (BI; cigarettes/day multiplied by number of years) of smokers in this study was from 12 to 60, our results suggest that even light smoking of less than 20 cigarettes/day for 3 years can result in the development of hearing loss of 12 kHz frequency in young adults. Binary logistic regression analysis again showed a correlation between hearing loss (≥40 dB of auditory thresholds in 12 kHz frequency) and light smoking (12 ≤ BI ≤ 60). Thus, this study showed that auditory threshold at 12 kHz frequency could be a sensitive marker for hearing in young adults. More importantly, we for the first time provided epidemiological evidence that light smoking might affect hearing level at 12 kHz frequency and revealed a new risk of light smoking.