HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/9/1165 02-0580fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ASANUMA, K. Articles by TOMINO, Y. Search for Related Content PubMed PubMed Citation Articles by ASANUMA, K. Articles by TOMINO, Y.

MAP-LC3, a promising autophagosomal marker, is processed during the differentiation and recovery of podocytes from PAN nephrosis¹

KATSUHIKO ASANUMA^*, ISEI TANIDA, ISAO SHIRATO^*, TAKASHI UENO, HISATSUGU TAKAHARA^*, TOMOHITO NISHITANI^*,, EIKI KOMINAMI and YASUHIKO TOMINO^*,2

^* Division of Nephrology, Departments of Internal Medicine and
Biochemistry, Juntendo University School of Medicine, Tokyo, Japan

²Correspondence: Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: yasu{at}med.juntendo.ac.jp

SPECIFIC AIMS

To better understand the role of LC3 processing in differentiated cells, we investigated the processing during the differentiation and recovery of podocytes from damage of puromycin aminonucleoside (PAN).

PRINCIPAL FINDINGS

1. LC3 is processed during the differentiation of podocytes
To investigate whether LC3-I, the cytosolic form of microtubule-associated protein 1 light chain 3 (LC3), is specifically processed to LC3-II (the membrane-bound form) with the differentiation of podocytes, we used MPC cells, which conditionally differentiate into podocytes. Under permissive conditions, MPC cells proliferate and maintain an epithelial phenotype with a cobblestone-like morphology (Fig. 1 A, a, c). LC3-I was abundant in the lysates of the undifferentiated MPC cells (Fig. 1B , lane 1). When MPC cells are shifted to nonpermissive conditions, they stop proliferating and begin to convert into arborized cells (Fig. 1A, b, d ). After differentiation, LC3-II was abundant in the lysates of the differentiated MPC cells and LC3-I levels decreased significantly (Fig. 1B , lane 2). A large amount of LC3-I is expressed in the cytosolic fraction and MS fraction of the undifferentiated cells (Fig. 1B , lanes 4, 5). When differentiation is induced, large amounts of LC3-II are found in the ML fraction and MS fraction (Fig. 1B , lanes 6, 7). For LC3 processing, Apg3p, an E2-like enzyme, and Apg7p, an E1-like enzyme, are both essential. Apg3p accumulates significantly in differentiated MPC cells compared with undifferentiated cells (Fig. 1C , lane 2 vs. lane 1). Concomitant with differentiation, LC3-I was processed to LC3-II in a matter of days, whereas LC3 processing in autophagy took just hours (Fig. 1E vs. D).

View larger version (48K): [in this window] [in a new window]   Figure 1. LC3-I is processed during the differentiation of MPC cells into podocytes. A) MPC cells differentiate into podocytes under nonpermissive conditions. When MPC cells were propagated under permissive conditions (a, c) (33°C, INF+), cells showing characteristic cobblestone morphology were observed by light microscopy (a). Little synaptopodin, a marker protein for podocytes, is recognized by immunofluorescent analysis using anti-synaptopodin antibody (c). When MPC cells were differentiated under nonpermissive conditions for 7 days (b, d) (37°C, INF-), differentiated and arborized cells were observed (b). The large, flat cells have developed prominent processes with spindle-like projections. Differentiated arborized MPC cells express synaptopodin, indicating that the cells differentiate into podocytes under these conditions (d). Bar, 25 µm. LC3-II accumulates in differentiated MPC cells. B) LC3-II accumulates in differentiated MPC cells. Lysates of undifferentiated (lane 1) and differentiated (lane 2) MPC cells were subjected to immunoblot analysis with anti-LC3 antibody. Cell homogenates from undifferentiated (lanes 3–5) and differentiated (lanes 6–8) MPC cells were fractionated into a mitochondrial lysosomal fraction (ML) (lanes 3, 6), microsomal fraction (MS) (lanes 4, 7), and cytosol fraction (Cyto) (lanes 5, 8). Arrows indicate LC3-I and LC3-II. C) Apg3p levels increase during the differentiation of MPC cells into podocytes. Lysates of undifferentiated and differentiated MPC cells were subjected to immunoblot analysis with anti-Apg3p antibody. Arrow indicates Apg3p. The Apg3p level is greater in differentiated MPC cells (lane 2) than undifferentiated cells (lane 1). D) LC3 processing in autophagy under starvation conditions. Undifferentiated MPC cells were cultured at 33°C for 1.5–5 h in Hanks’ solution with -interferon. After incubation, cells were subjected to immunoblot analysis using anti-LC3 antibody. E) LC3-I is processed to LC3-II during differentiation in a matter of days. MPC cells were induced to differentiate into podocytes.

2. LC3-II localized vesicles in differentiated MPC cells resemble autophagic vacuole-like structures
We next investigated the intracellular localization of LC3-II in differentiated MPC cells by indirect immunofluorescence microscopy. Before differentiation, LC3-I was distributed in the cytosol. When differentiation was induced, LC3-II was remarkably localized to punctate structures. The distribution of LC3-II in differentiated MPC cells was different from that of LAMP-1, a lysosomal marker, and that of M6PR, an endosomal marker. We investigated the localization of LC3 in differentiated MPC cells by immunoelectron microscopy using anti-LC3 antibody. LC3 localized membrane structures in differentiated MPC cells under nutrient-rich conditions resemble autophagic vacuoles in undifferentiated MPC cells under starvation conditions.

3. Differentiated MPC cells show little autophagic activity under nutrient-rich conditions
Formation of autophagic vacuoles is induced under starvation conditions but is repressed under nutrient-rich conditions. Whereas LC3-II localized vesicles in differentiated MPC cells resemble autophagic vacuoles, it is unlikely that autophagy is enhanced in differentiated MPC cells even under nutrient-rich conditions. We then investigated the autophagic activity in differentiated MPC cells under differentiation conditions. When autophagy occurs, LC3-II becomes associated with both the lumenal and cytosolic surfaces of the autophagosomal membrane and the lumenal LC3-II is degraded by lysosomal proteinases. The addition of E64d, a cysteine protease inhibitor, and pepstatin, an aspartic protease inhibitor, to the starvation medium inhibited lysosomal protein degradation, resulting in the accumulation of LC3-II. When differentiated MPC cells were cultured under differentiation conditions in the presence of these inhibitors for 24 h, no further accumulation of LC3-II was found compared with that in the absence of the inhibitors, indicating that LC3-II in differentiated MPC cells is not involved in lysosomal degradation.

4. The increase in LC3 in podocytes correlates with the recovery from damage due to PAN nephrosis
Considering that LC3-I is processed to LC3-II in parallel with podocyte differentiation, LC3-II may play an essential role in the physiological function of podocytes. It is known that the developmental sequence of podocytes is reversed in nephrotic syndrome: foot processes spread out and podocytes come together. We examined the effect of PAN on LC3-II in differentiated MPC cells. After differentiation of the MPC cells, PAN was added to the medium and the cells were incubated. After removing the reagent, differentiated MPC cells were incubated in the medium. A dynamic morphological change occurred in the differentiated MPC cells by days 2 and 3: a significant reduction in process formation, essential for the function of podocytes in normal kidney, and disarrangement of actin filaments (Fig. 2 A, f and I vs. c). Under these conditions, LC3 levels decreased and staining was dispersed in the cytosol, whereas LC3 was localized to punctate structures in untreated cells (Fig. 2A, d and g vs. a ). Seven days after PAN treatment, actin cytoskeleton arrangement and process formation of the cells had recovered their original appearance in control cells (Fig. 2A, k, l ). At this time, LC3-II was remarkably localized to punctate structures (Fig. 2A, j ) and the amount for LC3-II increased significantly (Fig. 2B , PAN 7 days).

View larger version (52K): [in this window] [in a new window]   Figure 2. Morphology of differentiated MPC cells and the distribution of LC3 in cells after treatment with PAN. A) Differentiated MPC cells were treated with 50 µg/mL of PAN for 24 h. After removal of the PAN, cells were further cultured for 6 days to recover from the damage induced by PAN. Cells were fixed on days 2 (d–f), 3 (g–i), and 7 (j–l) after treatment and permeabilized for immunofluorescence confocal microscopy using rhodamine-conjugated phalloidin (b, e, h, k) and anti-LC3 antibody (a, d, g, j). Cell morphology was observed by phase-contrast microscopy (c, f, i, l). Bar, 25 µm. Control cells show well-developed process formation and contain long bundles of actin filaments that terminate in the periphery of the cell processes (a–c). PAN treatment induced process retraction and cell rounding by 2-3 days (f, i) with a loss of actin filaments (e, h) (PAN 2 and 3 days). LC3 became dispersed and/or the level decreased (d, g). 6 days after removal of PAN, the process formation was recovered (l) and both actin filaments (k) and the dotted pattern of LC3-staining increased compared with control (PAN 7 days vs. control). B) LC3-II levels were significantly increased on day 7, closely correlated with morphological recovery of the PAN-damaged differentiated MPC cells. After damage to differentiated MPC cells by PAN, LC3-II levels were decreased on days 2 and 3 (PAN day 2 and 3), when the process formation was significantly reduced (A, f, i). On day 7, LC3-II levels were increased in accord with morphological recovery. Cell lysates (40 µg of total proteins per lane) were subjected to immunoblot analysis with anti-LC3 antibody. Graph shows data in arbitrary units for the density of the LC3 bands in each sample. Columns and bars represent the mean ± SE of the results of experiments performed in triplicate. *Difference relative to the control is significant at the 95% confidence level.

CONCLUSIONS

We found for the first time that LC3-I is processed to LC3-II according to differentiation and that the amount and intracellular localization of LC3-II show a significant correlation with both process formation in differentiated MPC cells and the recovery from damage of PAN. Whereas LC3-II localized vesicles in differentiated MPC cells resemble autophagosomes during starvation-induced autophagy, differentiated MPC cells show little autophagic activity under differentiation conditions. These results suggest that LC3 is processed during podocyte differentiation to show an important property related to the formation of podocytes.

It is surprising that LC3-I is processed to LC3-II with cellular differentiation, because the processing plays an indispensable role in the dynamics of membrane formation, especially in autophagy. It has been reported that autophagy occurs significantly in proximal tubular cells of the kidney, but there is no report of autophagy in podocytes. One possibility is that LC3-II localized vesicle play an indispensable role in the differentiation of podocytes (Fig. 3 ). During differentiation of the podocytes, LC3 is processed and localized to vesicles. When PAN was given to differentiated MPC cells, LC3-II dispersed according to the morphological changes of process formation. After recovery from damage due to PAN, LC3-II increased and localized to vesicles. An increase in LC3 was also observed upon recovery from nephrosis in PAN nephrosis model rats. We hypothesized that the LC3-II localized vesicles in podocytes have an essential role in podocytes different from autophagosomal function of autophagosomes during starvation-induced autophagy.

View larger version (32K): [in this window] [in a new window]   Figure 3. Working hypothesis of LC3-II localized vesicles in podocytes. During differentiation to podocytes, LC3-I is processed to LC3-II, and LC3-II localizes to vesicles. This process is similar to the formation of autophagic vacuoles during starvation-induced autophagy. Little LC3-II on the vesicles in podocytes is degraded by lysosomal proteases in lysosomes; LC3-II on autophagic vacuoles during starvation-induced autophagy is degraded by lysosomal proteases after fusion of autophagosomes to lysosomes. LC3-II localized vesicles in podocytes have a physiological function different from autophagic vacuoles during starvation-induced autophagy.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0580fje; to cite this article, use FASEB J. (April 22, 2003) 10.1096/fj.02-0580fje

This article has been cited by other articles:

				C. Chu and A. J. Shatkin Apoptosis and Autophagy Induction in Mammalian Cells by Small Interfering RNA Knockdown of mRNA Capping Enzymes Mol. Cell. Biol., October 1, 2008; 28(19): 5829 - 5836. [Abstract] [Full Text] [PDF]

				A. Nakajima, H. Kurihara, H. Yagita, K. Okumura, and H. Nakano Mitochondrial Extrusion through the Cytoplasmic Vacuoles during Cell Death J. Biol. Chem., August 29, 2008; 283(35): 24128 - 24135. [Abstract] [Full Text] [PDF]

				Q. Chen, W. Xie, D. J. Kuhn, P. M. Voorhees, A. Lopez-Girona, D. Mendy, L. G. Corral, V. P. Krenitsky, W. Xu, L. Moutouh-de Parseval, et al. Targeting the p27 E3 ligase SCFSkp2 results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy Blood, May 1, 2008; 111(9): 4690 - 4699. [Abstract] [Full Text] [PDF]

				C. Pecqueur, T. Bui, C. Gelly, J. Hauchard, C. Barbot, F. Bouillaud, D. Ricquier, B. Miroux, and C. B. Thompson Uncoupling protein-2 controls proliferation by promoting fatty acid oxidation and limiting glycolysis-derived pyruvate utilization FASEB J, January 1, 2008; 22(1): 9 - 18. [Abstract] [Full Text] [PDF]

				J.-h. Zhu, C. Horbinski, F. Guo, S. Watkins, Y. Uchiyama, and C. T. Chu Regulation of Autophagy by Extracellular Signal-Regulated Protein Kinases During 1-Methyl-4-Phenylpyridinium-Induced Cell Death Am. J. Pathol., January 1, 2007; 170(1): 75 - 86. [Abstract] [Full Text] [PDF]

				J. J. Jennings Jr., J.-h. Zhu, Y. Rbaibi, X. Luo, C. T. Chu, and K. Kiselyov Mitochondrial Aberrations in Mucolipidosis Type IV J. Biol. Chem., December 22, 2006; 281(51): 39041 - 39050. [Abstract] [Full Text] [PDF]

				J.-i. Iwata, J. Ezaki, M. Komatsu, S. Yokota, T. Ueno, I. Tanida, T. Chiba, K. Tanaka, and E. Kominami Excess Peroxisomes Are Degraded by Autophagic Machinery in Mammals J. Biol. Chem., February 17, 2006; 281(7): 4035 - 4041. [Abstract] [Full Text] [PDF]

				M. Koike, M. Shibata, S. Waguri, K. Yoshimura, I. Tanida, E. Kominami, T. Gotow, C. Peters, K. von Figura, N. Mizushima, et al. Participation of Autophagy in Storage of Lysosomes in Neurons from Mouse Models of Neuronal Ceroid-Lipofuscinoses (Batten Disease) Am. J. Pathol., December 1, 2005; 167(6): 1713 - 1728. [Abstract] [Full Text] [PDF]

				C. Yan, M. Tanaka, K. Sugie, T. Nobutoki, M. Woo, N. Murase, Y. Higuchi, S. Noguchi, I. Nonaka, Y. K. Hayashi, et al. A new congenital form of X-linked autophagic vacuolar myopathy Neurology, October 11, 2005; 65(7): 1132 - 1134. [Abstract] [Full Text] [PDF]

				W. H. Yu, A. M. Cuervo, A. Kumar, C. M. Peterhoff, S. D. Schmidt, J.-H. Lee, P. S. Mohan, M. Mercken, M. R. Farmery, L. O. Tjernberg, et al. Macroautophagy--a novel {beta}-amyloid peptide-generating pathway activated in Alzheimer's disease J. Cell Biol., October 10, 2005; 171(1): 87 - 98. [Abstract] [Full Text] [PDF]

				T. Kouno, M. Mizuguchi, I. Tanida, T. Ueno, T. Kanematsu, Y. Mori, H. Shinoda, M. Hirata, E. Kominami, and K. Kawano Solution Structure of Microtubule-associated Protein Light Chain 3 and Identification of Its Functional Subdomains J. Biol. Chem., July 1, 2005; 280(26): 24610 - 24617. [Abstract] [Full Text] [PDF]

				I. Tanida, T. Ueno, and E. Kominami Human Light Chain 3/MAP1LC3B Is Cleaved at Its Carboxyl-terminal Met121 to Expose Gly120 for Lipidation and Targeting to Autophagosomal Membranes J. Biol. Chem., November 12, 2004; 279(46): 47704 - 47710. [Abstract] [Full Text] [PDF]

				I. Tanida, Y.-s. Sou, J. Ezaki, N. Minematsu-Ikeguchi, T. Ueno, and E. Kominami HsAtg4B/HsApg4B/Autophagin-1 Cleaves the Carboxyl Termini of Three Human Atg8 Homologues and Delipidates Microtubule-associated Protein Light Chain 3- and GABAA Receptor-associated Protein-Phospholipid Conjugates J. Biol. Chem., August 27, 2004; 279(35): 36268 - 36276. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/9/1165 02-0580fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ASANUMA, K. Articles by TOMINO, Y. Search for Related Content PubMed PubMed Citation Articles by ASANUMA, K. Articles by TOMINO, Y.