Correlation between ZO proteins and cell cycle-dependent hypertrophy

include cardiac hypertrophy and compensatory nephrotic hypertrophy. Hypertrophy can be triggered by numerous factors, such as growth factors, hormones, Ca²⁺ signaling, integrin signaling and nutrient overabundance [269-272]. One kind of hypertrophy, resulting from cell cycle arrest at the G1/S phase, is classified as cell cycle-dependent hypertrophy [273].

Cell cycle-dependent hypertrophy results from increased cellular content of RNA and protein without DNA replication [274,275]. I observed a significant increase of cell volume following ZO-1 and ZO-2 silencing. Cell hypertrophy induced by ZO-1 depletion is mainly due to cell cycle inhibition at the G1/S phase, since ZO-1/p21 co-silencing was sufficient

to restore cell-cycle arrest and decreased cell size as well. On the other hand, ZO-3 silencing did not significantly alter cell cycle progression, yet increased p21 and CycD1 protein abundance to an even higher extent than ZO-1 silencing. Renal tubular cells exhibit both proliferative and hypertrophic responses following acute kidney injury (AKI) that is concomitant with elevated cyclins (cyclin D1, D3, A, B) and p21 induction [16,276].

D-type cyclins (D1, D2 and D3) are synthesized in the early G1 phase and are degraded as cells exit the G1 phase and enter the S phase [277]. Their activity is associated with renal hyperplasia and hypertrophy [274]. P21, a cyclin-dependent kinase inhibitor, is considered to be a negative regulator of D-type cyclins. Its activity is altered in response to a variety of intracellular and extracellular stress signals [278]. Under certain conditions, cytoplasmic p21 promotes cell survival through inhibition of apoptosis-related proteins [279]. P21 and CycD1 are constitutively expressed at low levels under physiological conditions (confluent mCCDcl1

cells also express low levels of p21 and CycD1). However, following AKI induced by ischemia, ureteral obstruction or cisplatin, p21, but not other cyclin kinase family members, is rapidly induced in mouse kidney cells, in the thick ascending limb of Henle and in the distal convoluted tubule [280]. Following either cisplatin administration or ischemia-reperfusion, p21-deficient mice displayed increased cell proliferation but developed severe morphological damage and had high rate of mortality as compared to wild-type littermates, indicating that p21 induction ameliorated AKI [281,282]. During AKI recovery, balance between p21 and CycD1 expression is probably important for appropriate cell-cycle and cell size regulation of renal tubular cells. Since loss of cell-cell contacts, and loss of ZO proteins, is an early event in ischemic AKI [31], ZO-1 and ZO-3 likely contribute to the regulation of p21 and CycD1

expression during this process.

Perspectives

In this work, I investigated roles of ZO proteins in regulating proliferation of mCCDcl1

cells, an in vitro model of renal CD principal cells. Future studies should focus on the roles of ZO proteins in regulating kidney function and proliferation in vivo. Although ZO-1 and ZO-2-deficient mice are embryonic lethal [150,166], adult ZO-2 chimera mice are viable [168]. Interestingly, gross anatomical and histological studies of adult ZO-2 chimera mice revealed major abnormalities in testis and kidney. While effects on testis have been described in some detail, the mechanisms of ZO-2-deficency on kidney abnormality have not yet been investigated. These ZO-2 chimera mice may provide a good in vivo model for investing ZO proteins in regulating kidney function and proliferation. In addition, a previous study showed that ZO-3 knockout mice are viable and lack an obvious phenotype under normal laboratory conditions [113]. Whether these animals exhibit abnormalities under stress conditions, such as AKI, is unclear. Examination of renal cell proliferation following AKI in conditional ZO kidney-specific knockout mice that display genetically-labeled -galactosidase tubular epithelial, but not interstitial, cells would help resolve the roles of ZO proteins in regulating kidney function and proliferation.

VIII Addendum

Publication Ⅱ

I participated in a study led by my mentor Dr Hasler that demonstrated induction of autophagy by hypertonicity and how this helps protect cells from death. I analyzed the effects of rapamycin and ATG12 knockdown, which both modulate autophagic activity, on hypertonicity-induced cell death, and drafted Figure 2.

©2013 Landes Bioscience. Do not distribute.

BAsic ReseARch PAPeR

Introduction

The maintenance of intracellular isotonicity is a fundamental requisite for cell survival. While the kidney protects mamma-lian cells from large osmotic changes, numerous cell types are

Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, Lc3-ii generation and Ptdlns3K-dependent formation of Lc3- and ATG12-positive puncta. Lysosomotropic agents chloroquine and bafilomycin A₁, but not nutrient deprivation or rapamycin treatment, further increased Lc3-ii generation, as well as ATG12-positive puncta, indicating that hypertonic stress increases autophagic flux. Autophagy induction upon hypertonic stress enhanced cell survival since cell death was increased by ATG12 siRNA-mediated knockdown and reduced by rapamycin. We additionally showed that hypertonicity induces fast reorganization of microtubule networks, which is associated with strong reorganization of microtubules at centrosomes and fragmentation of Golgi ribbons. Microtubule remodeling was associated with pericentrosomal clustering of ATG12-positive autolysosomes that colocalized with sQsTM1/p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is at least partly selective. efficient autophagy by hypertonic stress required microtubule remodeling and was DYNc/

dynein-dependent as autophagosome clustering was enhanced by paclitaxel-induced microtubule stabilization and was reduced by nocodazole-induced tubulin depolymerization as well as chemical (ehNA) or genetic [DcTN2/dynactin 2 (p50) overexpression] interference of DYNc activity. The data document a general and hitherto overlooked mechanism, where autophagy and microtubule remodeling play prominent roles in the osmoprotective response.

Hypertonic stress promotes autophagy and microtubule-dependent

autophagosomal clusters

Paula Nunes,^1,3 Thomas ernandez,^1,2,† isabelle Roth,^1,2,† Xiaomu Qiao,^1,2 Déborah strebel,^1,2 Richard Bouley,³ Anne charollais,¹ Pierluigi Ramadori,¹ Michelangelo Foti,¹ Paolo Meda,¹ eric Féraille,^1,2,‡ Dennis Brown^3,‡ and Udo hasler^1,2,3,*

1Department of cellular Physiology and Metabolism; University of Geneva; Geneva, switzerland; ²service of Nephrology; Department of Medical specialties;

University of Geneva; Geneva, switzerland; ³center for systems Biology; Program in Membrane Biology and Division of Nephrology; Massachusetts General hospital;

Boston, MA UsA

†These first authors contributed equally to this work.

‡These last authors contributed equally to this work.

Keywords: ATG12, Golgi apparatus, MAP1LC3/LC3, MAPRE1/EB1, MTOC, SQSTM1/p62, apoptosis, autophagy, dynein, lysosome, microtubule, osmotic stress, proteasome, rapamycin, tubulin, ubiquitin

Abbreviations: ACTB, β-actin; ANXA5, annexin A5; ATG12, autophagy-related 12; DCTN, dynactin; DYNC, dynein;

EB, end-binding protein; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine; ER, endoplasmic reticulum; GA, Golgi apparatus;

GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; KIF, kinesin; LAMP, lysosome-associated membrane protein; LC3B/MAP1LC3B, microtubule-associated protein 1 light chain 3 beta; LDH, lactate dehydrogenase;

MAPRE1 (also known as end-binding protein 1, EB1), microtubule-associated protein RP/EB family member 1; MT, microtubule;

MTOC, microtubule-organizing center; MTORC, mechanistic target of rapamycin complex; Ptdlns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol 3-phosphate; RVI, regulatory volume increase; siRNA, small interfering RNA;

SQSTM1 (also known as p62), sequestosome 1; TUBA, α-tubulin; TUBG, γ-tubulin; UPS, ubiquitin proteasome system

exposed to aniosmotic environments in normal daily functions.

These include epithelial and interstitial cells of the renal medulla, blood cells passing through the vasa recta, and epithelial cells of the small intestine.¹ In pathophysiological conditions such as hypernatremia and diabetes cells throughout the body are

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BAsic ReseARch PAPeR BAsic ReseARch PAPeR

space, cell compression by hypertonic stress may interfere with the organization of microtubule (MT) networks and intracellular sorting, processes that are required for autophagic clearance of protein aggregates.^28-30 Thus, to understand how autophagy oper-ates in the context of changing cell volume, a better understand-ing of how osmotic compression affects MT networks is required.

Indeed, surprisingly little is known on the effects of hypertonic stress on MT structure and behavior.⁸ MT remodeling is a hall-mark response to many different types of cellular stresses, includ-ing ischemia,^31,32 oxidative stresses,³³ cold and heat shock,^34-37 as well as ionizing³⁸ and UV irradiation.³⁹ It stands to reason that MT remodeling may also occur during hypertonic stress, and that this remodeling could affect autophagy.4,7,8,28,29

In the present study, we demonstrate that in mammalian cells autophagosomes accumulate shortly following hypertonic chal-lenge as a result of increased autophagic flux and that autophagy is paramount to an effective osmoprotective response. We fur-ther demonstrate that MT networks are reorganized immediately following challenge and that this is associated with strong MT reorganization at centrosomes. We show that both MT reorgani-zation and DYNC/dynein are required for efficient formation of autolysosomal clusters in a pericentrosomal compartment. Our findings not only provide new insights into mechanisms under-lying osmotic adaptation, but also show that hypertonic insult may serve as a new, easily accessible and physiologically relevant model for the study of autophagy.

Results

Hypertonic stress increases autophagic flux that enhances cell survival. We have previously shown that hypertonic stress alters intracellular membrane trafficking in LLC-PK₁ renal proximal tubule-like cells.²⁵ In the present study, using the same cell line, we compared time-dependent variations of cell size with pro-tein degradation and examined the possibility that such changes are accompanied by increased autophagic activity. As expected, reduced cell volume by hypertonic challenge was immediately followed by regulatory volume increase (RVI), as reflected by half-maximal recovery time of cell volume (R_vt_1/2) and steady-state recovered volume (R_vmax) values (Fig. 1A). As revealed by pulse-chase assays, hypertonic stress increased the degrada-tion of long-lived protein to an extent similar to that induced by nutrient deprivation, a commonly used model of cellular stress (Fig. 1B). Elevated protein damage was, in turn, accompanied by increased autophagic activity, as revealed by increased cleavage and lipidation of microtubule-associated protein 1 light chain 3 β (referred here as LC3-I) to LC3-II, a bona fide autophagy marker (Fig. 1C). The extent of LC3-II generation by hypertonic stress was similar to that induced by nutrient deprivation. Increased LC3-II production by either source of stress was not additive (Fig. 1C). As revealed by confocal microscopy, both nutri-ent deprivation and hypertonic stress increased the number of puncta positive for both LC3 and ATG12, another auto-phagosome marker (Fig. 1D). Our data are in agreement with exposed to osmotic stress. Moreover, some treatments for diseases

such as hyponatremia, cystic fibrosis and hemorrhagic shock involve administration of hypertonic fluids.^2-4 Thus understand-ing the consequences of osmotic shock on cellular structure and functions as well as the mechanisms underlying osmoprotective responses are relevant not only for understanding basic cellular function but also have clinical implications.

Because of passive water diffusion across the plasma mem-brane, even small increases in extracellular membrane-imper-meable solutes induce cell shrinkage, molecular crowding and elevate intracellular ionic strength.^5-7 Cell survival depends on rapid retrieval of lost water, which occurs by the accumula-tion of electrolytes from outside the cell through the process of regulatory volume increase (RVI). Failure of osmoprotective mechanisms leads to apoptosis.⁸ Despite RVI, accumulation of inorganic ions and molecular crowding together destabilize pro-tein secondary structure. Propro-tein damage and aggregation that occurs upon hypertonic exposure, however, has come to light only in recent years.^7,9-11 While genes controlling the recruitment of endosomal sorting complexes and lysosomes have been identi-fied as playing a prominent role in reducing protein aggregation and cell death by hypertonic stress,¹² the mechanisms that drive protein degradation remain poorly understood.

Degradation of misfolded and damaged proteins is performed principally by the ubiquitin proteasome system (UPS) and autophagy. During UPS-mediated proteolysis, ubiquitination targets proteins for destruction by the proteasome.¹³ This is a selective process responsible for the degradation of abnormal pro-teins and normal propro-teins with short half-lives. Macroautophagy (referred to here as autophagy) is a catabolic pathway responsible for the degradation of long-lived proteins, macromolecular aggre-gates and damaged cytoplasmic organelles, whereby the material targeted for destruction is sequestered within membrane-bound organelles (autophagosomes) that fuse with lysosomes to gain proteolytic properties.^14-17 The importance of autophagic removal of damaged proteins is illustrated by the prominent conse-quences of its dysregulation in neurodegenerative diseases such as Alzheimer and Parkinson disease.^18,19 Although long viewed as a random, nonselective, degradation system, recent evidence has uncovered different types of selective autophagic degradation pathways where protein ubiquitination plays a prominent role.^20,21 Besides ubiquitin itself, the ubiquitin-binding protein SQSTM1 (sequestosome 1) plays a key role in the elimination of ubiquiti-nated protein aggregates by promoting aggregate formation prior to their incorporation into autophagosomes.²² Protein aggregates that form upon hypertonic exposure are undoubtedly eliminated in order to ensure cell survival. However, whether autophagy, selective or nonselective, plays a role in clearing damaged protein as a part of the osmoprotective response to hypertonic stress has not been addressed.

We and others have previously demonstrated altered vesi-cle trafficking and sorting shortly following hypertonic chal-lenge,^23-26 which may impinge on the cell’s ability to cope with protein damage. A universal consequence of water efflux is that

increased cell death by siRNA against ATG12, hypertonicity-induced cell death was decreased when cells were pretreated with rapamycin, as revealed by flow cytometry (Fig. 2C) and phase contrast microscopy (Fig. 2D). Collectively, these findings indi-cate that increased autophagic flux by hypertonic stress helps pro-mote cell survival.

Hypertonic stress induces perinuclear clustering of autolyso-somes containing sequestered SQSTM1. Increased autophagic flux by hypertonic stress was associated with perinuclear cluster-ing of LC3- and ATG12-positive puncta (Fig. 1D and E). We examined the nature of these clusters in more detail (Fig. 3).

While RFP-LC3 puncta were readily visible, quantification was unreliable due to variations of transfection efficiency and ensuing intercellular heterogeneity. In contrast, IgG against endogenous ATG12 produced a signal that was substantially more homog-enous. Moreover, a recent study has proposed that ATG12–

ATG5 complexes are present in autolysosomes.⁴³ We therefore used ATG12 as a means to accurately quantify autophagosome perinuclear clustering. Time-course experiments revealed that while not apparent immediately following challenge (≤ 2 min), ATG12-positive puncta transiently increased in size after longer periods of time (Fig. 3A) with a maximal effect achieved 30 min following challenge. Their appearance was abolished in cells transfected with siRNA against ATG12 and in cells pretreated with LY-294002, an inhibitor of phosphatidylinositol-3-kinase (Ptdlns3K), a key component of classic autophagy¹⁴ (Fig. 3A;

Fig. S1). Similar proliferation of ATG12-positive puncta was induced by equiosmolar mannitol (Fig. 3A) but not urea (not shown), indicating that their accumulation arises from cell shrinkage following hypertonic challenge. Both the number and size of structures returned toward basal levels after sustained challenge (8 h and 24 h, Fig. 3A). This was accompanied by decreased steady-state levels of LC3-II (Fig. 3B) suggesting that while autophagic flux is particularly high upon hyper-tonic challenge, it subsides after longer periods of time, well after RVI (Fig. 1A), possibly reflecting cell adaptation. Close inspection of perinuclear clusters revealed good colocalization between ATG12 and lysosome-associated membrane protein LAMP1, a late endosomal/lysosomal marker (Fig. 3C). Similar to differences between hypertonic stress and nutrient deprivation (Fig. 1D), confocal microscopy analysis revealed that ATG12-positive puncta were significantly larger upon hypertonic chal-lenge than following rapamycin chalchal-lenge, both in the absence or presence of chloroquine or bafilomycin A₁ (Fig. 3D and F).

The number of hypertonicity-challenged cells displaying large, of GFP-LC3 puncta in response to hypertonic stress.⁴⁰ A

strik-ing difference between nutrient deprivation and hypertonic stress in LLC-PK₁ cells was the appearance of large apical, perinuclear structures induced by hypertonic stress that were more apparent than that following nutrient deprivation (Fig. 1D, arrows and enlarged images). ATG12 perinuclear accumulation by hyperto-nicity was also observed in both primary human monocytes and macrophages (Fig. 1E), suggesting that this observation may be extended to other cell types.

Similar to nutrient deprivation, induction of LC3-II genera-tion by hypertonic stress was not additive to that induced by rapamycin, an activator of autophagy that inhibits the autoph-agy-inhibitory MTORC1 protein kinase complex and its analogs (Fig. 1F), suggesting that mechanisms responsible for LC3-I lipidation by hypertonicity share common players with nutri-ent deprivation and rapamycin. We next examined whether autophagosome accumulation by hypertonicity results from increased autophagic flux or rather suppression of downstream stages of the autophagy pathway that lead to the accumulation of autolysosomes. Increased LC3-II generation by stimuli that increase autophagic flux is further increased by chloroquine and bafilomycin A₁,^41,42 which block endosomal/lysosomal acidification. Increased LC3-II immunoreactivity by hyperto-nicity was further increased by treating cells with either agent (Fig. 1F). Increased long-lived protein degradation by hyperto-nicity (Fig. 1B), together with the observation that conversion of LC3-I to LC3-II in hypertonicity-challenged cells was not increased by either nutrient deprivation or rapamycin (Fig. 1C and F) but was enhanced by lysosomotropic agents (Fig. 1F), indicated that hypertonic stress increases autophagic flux.

The contribution of autophagy to cell survival under condi-tions of hypertonic stress was next examined (Fig. 2). Apoptosis can be triggered when cell survival mechanisms induced by hypertonicity are impaired.⁵ The relevance of autophagy in pro-tecting cells against hypertonic-induced cell death was exam-ined by flow cytometry and by measuring lactate dehydrogenase (LDH) release. As revealed by both methods, cell death under hypertonic conditions was increased in cells transfected with siRNA against ATG12, as compared with cells transfected with scrambled siRNA (Fig. 2A). Similarly, increased cell death was associated with increased vacuolization and increased numbers of detached cells, after prolonged periods of hypertonic chal-lenge (Fig. 2B). The efficiency of siRNA in reducing ATG12 expression is depicted in Figure S1. We next examined the effects of rapamycin on hypertonicity-induced cell death. Mirroring

Figure 1 (See opposite page). hypertonic stress increases autophagic flux. (A) Variations of LLc-PK₁ cell volume in cells continuously exposed to isos-motic medium or challenged with Nacl (350 and 500 mOsmol/kg). The black arrow indicates when cells were challenged. half-maximal recovery time of cell volume (R_vt_1/2) and steady-state recovered volume (R_vmax) by each challenge is shown at right. (B) Protein degradation by nutrient deprivation or Nacl-hypertonic stress (500 mOsmol/kg) was quantified as [³h]L-valine released in the cell media. (C) Lc3-i conversion to Lc3-ii in nutrient-starved cells, cells challenged with Nacl or cells subjected to both stimuli. Quantification of Lc3-ii levels is shown on the right. (D) confocal z-stacks illustrating increased number of ATG12-positive puncta after 1 h of nutrient deprivation or 30 min of Nacl challenge. White arrows depict large, apical particles upon Nacl challenge. (E) confocal z-stacks depicting the formation of perinuclear ATG12-positive puncta in primary human monocytes and differenti-ated macrophages challenged with Nacl (400 mOsmol/kg) for 30 min. (F) immunoblot at left depicts Lc3-i conversion to Lc3-ii in cells challenged with Nacl (500 mOsmol/kg) for 30 min, rapamycin (Rapa; 200 nM) for 24 h or rapamycin and then Nacl. immunoblot at right depicts Lc3-i

from their accumulation and fusion in a constrained region of the cell. Several studies have suggested that inhibition of protea-some activity induces an accumulation of proteins that become substrates for autophagy.^45-47 While proteasome inhibition by MG132 or lactacystin alone did not induce ATG12-positive peri-nuclear clustering, both agents further increased their number and size following hypertonic challenge (Fig. 3D–F). Together, these data suggest that large, perinuclear ATG12-positive puncta observed upon hypertonic challenge principally consist of

Dans le document Role of Zonula Occludens (ZO) proteins in regulating kidney collecting duct principal cell proliferation and adhesion (Page 106-144)

Correlation between ZO proteins and cell cycle-dependent hypertrophy

VIII Addendum

©2013 Landes Bioscience. Do not distribute.

Hypertonic stress promotes autophagy and microtubule-dependent

autophagosomal clusters

©2013 Landes Bioscience. Do not distribute.

©2013 Landes Bioscience. Do not distribute.

conver-©2013 Landes Bioscience. Do not distribute.

©2013 Landes Bioscience. Do not distribute.