TGF-β1 Protects against Mesangial Cell Apoptosis via Induction of Autophagy^*

Introduction

In glomerular injury, counterbalancing of mesangial cell death and cell survival plays an important role in the pathogenesis of progressive glomerulosclerosis. Macroautophagy, more commonly referred to as autophagy, is a fundamental cellular homeostatic process that cells use to degrade and recycle cellular proteins and remove damaged organelles, and its role has been implicated in the pathogenesis of some disease states including cancer and neurodegeneration (1–3). The process of autophagy involves the formation of double-membrane-bound vesicles called autophagosomes that envelope and sequester cytoplasmic components, including macromolecular aggregates and cellular organelles for bulk degradation by a lysosomal degradative pathway (4). A number of evolutionarily conserved autophagy-related (Atg) proteins have been identified to date. The mammalian homolog of Atg8, known as microtubule-associated protein 1 light chain 3 (LC3),³ functions at least in part as a structural component in the formation of autophagosomes (5). The best characterized form LC3B (hereafter referred to as LC3) is the most widely used autophagic marker (6). Autophagosomes fuse with lysosomes to form autolysosomes, in which the sequestered contents are degraded by lysosomal hydrolases into their basic components, such as amino acids and fatty acids, which are returned to the cytosol for recycling (7, 8).

Autophagy represents a genetically programmed process that is evolutionarily conserved in eukaryotes from yeast to mammal and has important roles in various biological events such as cellular remodeling during development and differentiation and adaptation to changing environmental conditions and lifespan extension (1). Excessive autophagic activity can lead to type II programmed cell death that is morphologically distinct from apoptosis or type I programmed cell death and thereby contribute to the pathology of diseases (9). However, an emerging body of evidence supports the notion that autophagy can promote cell survival, and the accumulation of autophagosomes may signify a survival response intended to rid cells of misfolded proteins or damaged organelles (10, 11). Indeed, autophagy is implicated to play a protective role against the progression of certain human diseases, including cancer, muscular disorders, and neurodegeneration, such as Huntington, Alzheimer, and Parkinson diseases (12, 13). Autophagy is also implicated in cellular defense mechanism to prevent infection by certain pathogenic bacteria and viruses (13–15). To date, the role of autophagy in the kidney remains largely under-investigated. The potential protective role of autophagy as a survival mechanism after cytotoxic injury to the kidney is suggested by a recent report showing that autophagy decreases apoptosis of renal tubular cells induced by cisplatin (16). Thus, autophagy may act as a major cytoprotective pathway promoting cell survival and play an essential function for tissue homeostasis.

Transforming growth factor-β1 (TGF-β1) is a key mediator of glomerular injury response in progressive kidney diseases and regulates both cell growth and cell death. TGF-β1 acts as a multifunctional cytokine that regulates a number of biological responses including chemotaxis, cell cycle progression, differentiation, and apoptosis of target cells in a context- and cell-specific manner (17, 18). TGF-β1 has been shown to be a potent modulator of apoptosis in a variety of cell types, including epithelial cells, hepatocytes, hematopoietic cells, and lymphocytes (4, 19–21). The growth-inhibitory/pro-apoptotic effects of TGF-β1 have been well demonstrated in various cell types, including immune cells (22) and epithelial cells (23), consistent with its anti-inflammatory and tumor-suppressive functions. However, substantial evidence suggests that TGF-β1 also possesses the ability to inhibit apoptosis in some cell types, such as murine macrophages (24), human lung carcinoma cells (25), fibroblasts, myofibroblasts (5, 26–28), murine neurons (29), lung mesenchymal cells (30), and pulmonary artery endothelial cells (31). The effect of TGF-β1 on apoptosis and autophagy in primary mesangial cells has not been previously examined.

The Smad pathway represents a major signal transduction pathway activated by TGF-β superfamily ligands. Upon TGF-β1 stimulation, receptor-regulated (R)-Smads (Smad2 and Smad3) are activated and form complexes with the common mediator, Smad4. The R-Smad-Smad4 complex then translocates to the nucleus and functions as a transcriptional transactivator to regulate gene expression (32–34). In addition to the Smad pathway, there is a growing body of evidence that TGF-β1 signals through the activation of non-Smad signaling proteins, including TGF-β-activated kinase 1 (TAK1) and phosphatidylinositol 3-kinase (PI3K), and that there is a high degree of cell-type specificity of TGF-β1 signals (33, 35, 36). TAK1 triggers the activation of several downstream cell signaling cascades, including the MKK4/7-JNK and the MKK3/6-p38 MAPK (37, 38). We have previously reported that, in primary mouse mesangial cells (MMC), TAK1 mediates TGF-β1-induced activation of the MKK3-p38 signaling pathway (39, 40). Recent studies have also shown that TAK1 serves as an upstream activator of the PI3K-Akt pathway in hepatic macrophages and in osteoclasts (41, 42) and that TAK1-mediated Akt activation is essential for TGF-β1 to promote osteoclast survival (42). The PI3K-Akt pathway regulates a number of cellular processes including cell cycle progression and apoptosis (43). Cell cycle progression is controlled by cyclin/cyclin-dependent kinase (CDK) complexes and is negatively regulated by CDK inhibitors. Recent studies suggest that the PI3K-Akt pathway positively regulates G₁/S cell cycle progression through increased cyclin D1 by inhibition of proteolytic degradation (44) and decreased p27^KIP1 (p27) levels through ubiquitination-dependent proteolysis (45). The function and stability of p27 are regulated by its phosphorylation, and the main phosphorylation site on p27 is Ser-10. In early G₁ phase, Ser-10 phosphorylation is increased and allows exportin CRM1 (chromosome region maintenance 1)-dependent export and KIP1 ubiquitylation-promoting complex-mediated proteolysis of cytoplasmic p27 (46).

The present study was undertaken to determine the functional role of TGF-β1 in the induction of autophagy for mesangial cell survival and protection from apoptosis induced by serum deprivation. We demonstrate that treatment with TGF-β1 resulted in the induction of LC3 protein levels and punctate distribution of the autophagic vesicle-associated-form LC3-II, whereas suppressing the serum deprivation-induced caspase 3 activation and apoptosis in MMC. Moreover, we show that TGF-β1 induces autophagy via TAK1 and the PI3K-Akt dependent pathway. TGF-β1 failed to rescue the MMC from serum deprivation-induced apoptosis upon inhibition of autophagy by knockdown of LC3 by siRNA or by LC3 gene deletion. Our findings demonstrate the anti-apoptotic effects of TGF-β1 to rescue MMC from serum deprivation-induced apoptosis via induction of autophagy and promote mesangial cell survival.

MATERIALS AND METHODS

RESULTS

DISCUSSION

The balance between cell proliferation and cell death is central to many physiological processes, and its deregulation may induce disease. There is an emerging body of evidence indicating that autophagy is a major programmed mechanism controlling cell death and cell survival and is implicated in playing essential roles in cellular homeostatic process and, more recently, in the pathogenesis of disease states. TGF-β1 is a multifunctional cytokine that regulates cell proliferation as well as apoptosis and is a key mediator of glomerular injury response in progressive kidney diseases. Although its anti-proliferative and pro-apoptotic effects have been well demonstrated, TGF-β1 can also stimulate cell proliferation and exert anti-apoptotic effects, depending on the cell type or circumstances (56, 57). The underlying mechanisms induced by TGF-β1 to regulate cell fate remain poorly understood. In the present study we demonstrate that TGF-β1 prevents glomerular mesangial cells from undergoing apoptosis under serum deprivation conditions through activation of the PI3K-Akt-mTOR-S6K signaling axis and induction of autophagic protein LC3 and cell cycle progression through regulating the protein levels of cyclins and CDK inhibitor p27 to promote mesangial cell survival.

It is generally thought that the anti-proliferative effects of TGF-β1 are mediated through repressing the transcription of CDK genes as well as inhibiting CDK activities associated with G₁ to S phase progression through CDK inhibitors, resulting in cell growth arrest (58). Under serum deprivation conditions, however, our data provide evidence that TGF-β1 not only attenuates apoptosis of MMC but also promotes cell proliferation by inducing cell cycle progression rather than cell growth arrest. Serum deprivation severely reduced cell viability (Fig. 1A) through the induction of apoptosis, as indicated by caspase 3 activation (Fig. 1B), and increases in cells with hypodiploid apoptotic nuclei (Fig. 1C). We show that TGF-β1 treatment at the start of serum deprivation ameliorated these effects induced by serum deprivation. On the other hand, TGF-β1 was unable to inhibit caspase 3 activation if TGF-β1 treatment was delayed by its application 24 h after the start of serum deprivation (Fig. 1D). Thus, these data suggest that the protective effects of TGF-β1 against apoptosis by inhibiting caspase 3 activation might be mediated by affecting the early events in the apoptotic pathway. In addition to these anti-apoptotic effects of TGF-β1, our findings also provide evidence that under serum deprivation conditions TGF-β1 may promote MMC proliferation through the induction of cell cycle progression. It has been previously reported that in renal epithelial cells, mesangial cells, and several other cell types, TGF-β1 does not significantly alter cyclin-CDK levels (59–62) but, rather, inhibits the activity of CDKs through the kinase inhibitory protein (KIP) family of CDK inhibitors such as p27 and p21^CIP1 (CIP, cyclin inhibitory protein) (p21) (59). However, our data show that TGF-β1 attenuated serum deprivation-induced reduction of cyclin D1 and cyclin E protein levels (Fig. 2A). Furthermore, serum deprivation increased not only the protein level of p27 (middle panel of Fig. 2B) but also the phosphorylation level of p27 at Ser-10 (upper panel of Fig. 2B), whereas these increases were abrogated by TGF-β1 treatment. Although both p27 and p21 have been previously shown to be regulated by TGF-β1, in quiescent mesangial cells p27 is constitutively expressed, whereas p21 is barely detected (63, 64). Similar differential expression of p27 and p21 is also observed in normal rat kidneys. Moreover, the onset of mesangial cell proliferation in the anti-Thy1.1 model was associated with a reduction of p27 levels (64), suggesting that p27 might be a predominant regulator of MMC proliferation. In this context, our data indicate that TGF-β1 promotes MMC proliferation under serum deprivation conditions through retaining the levels of cyclins and reducing the cyclin-dependent kinase inhibitor p27.

Autophagy constitutes a degradative mechanism for the removal and turnover of bulk cytoplasmic constituents via the endosomal-lysosomal system. During autophagy, the formation of autophagosomes involves redistribution of LC3 from the cytosol to autophagic vesicles with conversion of cytosolic LC3-I to phospholipid conjugate form LC3-II and is widely used to monitor autophagy (48). Our data demonstrate that LC3-II levels were increased during the early period (within 1 h) of serum deprivation in MMC, but thereafter the levels of both LC3-I and LC3-II were dramatically reduced under prolonged serum deprivation conditions (Fig. 3, A and B). TGF-β1 treatment, however, resulted in increased LC3-I and LC3-II levels compared with the untreated cells under serum deprivation, as assessed by Western blotting (Fig. 3, A and B) and increased immunofluorescence staining of LC3 and the formation of autophagosomes indicated by punctate distribution of LC3 (Fig. 3C). We observed that the degree of increases in LC3-II protein levels was less than that of LC3-I protein with TGF-β1 treatment. There are two plausible explanations. First, it is possible that the influence of TGF-β1 stimulation with respect to the induction of LC3-I expression may be greater than its effect on the conjugation events after cleavage of LC3-I by Atg4, a cysteine protease responsible for the cleavage of the carboxyl terminus of LC3, to produce LC3-II (65) and thereby resulting in higher levels of LC3-I. Second, the increased autophagic flux by TGF-β1 stimulation subsequently would lead to rapid degradation of LC3-II itself that is bound to autophagosomes, therefore, resulting in the observed relatively lower levels of LC3-II protein by immunoblotting (50). We found that treatment with bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, and TGF-β1 resulted in greater accumulation of LC3-II (Fig. 3D), indicating that the observed lower level of LC3-II (Fig. 3B) is likely from rapid degradation of LC3-II by TGF-β1 induced-autophagy. Recent reports have shown evidence for transcriptional regulation of certain autophagy genes, including LC3 and Atg12, in the induction of autophagy (66, 67). In our study, TGF-β1 increased LC3 mRNA expression during serum deprivation in MMC, demonstrating that TGF-β1 induces LC3 at the transcriptional level, and that this is accompanied by increases in both LC3-I and LC3-II protein expression (Fig. 3E). Moreover, our data demonstrate that in MMC the induction of LC3 expression by TGF-β1 is mediated by non-Smad signaling pathways, namely TAK1 (Fig. 4, C and D) and PI3K-Akt (Fig. 5, B and C) and is independent of the Smad signaling pathway (Fig. 4, A and B).

Few studies have previously reported that TGF-β1 induces autophagy in bovine mammary gland epithelial cells and neonatal piglet gut epithelium in the context that autophagy represents type II programmed cell death, which is complementary to apoptosis type of cell death induced by TGF-β1 treatment (68, 69). However, mechanisms for activation of autophagy by TGF-β1 and its role in renal cell growth and cell death have not been elucidated. Our data provide the first evidence that TGF-β1-induced autophagy plays a crucial role in TGF-β1-mediated mesangial cell survival under serum deprivation condition. Knockdown of LC3 in wild-type MMC by specific siRNA (Fig. 6A) or LC3 gene deletion in MMC from LC3 null (LC3^−/−) mice (Fig. 6B) exhibited much higher caspase 3 activation compared with normal LC3 background under serum deprivation condition. More importantly, the protective effects of TGF-β1 against caspase 3 activation were drastically abrogated when LC3 expression was blocked. Furthermore, inhibition of autophagy by using bafilomycin A1 resulted in enhanced apoptosis and abrogated the protective effects of TGF-β1 against serum deprivation-induced apoptosis of MMC (Fig. 6C). Thus, our findings suggest that, at least in part, TGF-β1-induced autophagy mediates the protective effects of TGF-β1 against serum deprivation-induced mesangial cell apoptosis.

We and others have demonstrated that TGF-β1 activates Akt in various cell types including MMC (70). In MMC, we found that serum deprivation rapidly reduced phosphorylated Akt, whereas TGF-β1 stimulation up-regulated Akt phosphorylation in a time-dependent manner (Fig. 5A). Recent studies have reported that activation of Akt regulates the expression and phosphorylation of p27 (71, 72). In glioblastoma tumor cells, activated PI3K and elevated phosphorylated Akt levels were associated with reduced p27 levels, and inhibition of the PI3K-Akt pathway, either by a selective chemical inhibitor LY294002 or the expression of a kinase dead dominant-negative mutant of Akt, resulted in up-regulation of p27 and down-regulation of tumorigenicity, suggesting that enhanced cell viability occurred in part by down-regulation of p27 through activation of the PI3K-Akt pathway (72). Akt can also phosphorylate p27 and impair p27 function by preventing its nuclear import, leading to accumulation of cytoplasmic p27, and confers resistance to G₁ arrest in human breast cancer (71). Accordingly, our findings of increased levels of p27 protein as well as phosphorylation of p27 by serum deprivation in MMC may be caused by the deactivation of Akt, and conversely, TGF-β1 activates Akt and down-regulates p27 to promote cell cycle progression and mesangial cell survival.

Blockade of the PI3K pathway by its selective inhibitor LY294002 abrogated the activation of the PI3K-Akt-mTOR-S6K signaling axis, resulting in increased caspase 3 activation and reduction of LC3 expression regardless of TGF-β1 stimulation (Fig. 5B). Although we cannot rule out the potential negative effects of LY294002 on the expression of LC3 through the inhibition of class III PI3K complex (Vps34-Vps15-Beclin-1), known to be an autophagy inducer, our data indicate that the PI3K-Akt-mTOR-S6K signaling axis plays a pivotal role in TGF-β1-induced up-regulation of LC3 expression and inhibition of caspase 3 activation. We further confirmed our findings by specific blockade of Akt employing the expression of a dominant-negative mutant of Akt, which shows similar effects on LC3 expression and caspase 3 activation as treatment with LY294002 (Fig. 5C). Previous reports have shown that, in general, Akt activation inhibits autophagy through mTOR activation in various cell types (73–76), whereas our data suggest that in the context of serum deprivation conditions, TGF-β1-induced Akt activation induces autophagy in MMC. Indeed, our findings are supported by the more recent studies demonstrating that PI3K-Akt pathway can also positively regulate autophagy in HeLa cells (77) and NB4 promyelocytic leukemia cells (78). In addition, it has been previously reported that mTOR-S6K positively regulates 6-thioguanine-induced autophagy (79). Interestingly, a recent report by Hu et al. (80) indicates that PI3K-dependent induction of autophagy is also a tool used by the eukaryotic pathogen Cryptococcus neoformans to ensure survival and effect host cell damage during infection.

The cyclin-dependent kinase inhibitor p27 has been shown to be a critical link between induction of autophagy and inhibition of apoptosis in response to nutrient depletion (81). However, the mechanisms implicated in linking cell cycle progression and autophagy regulation are not yet known. We have shown that TGF-β1 stimulation strongly reduces p27 levels in wild-type MMC. Ubiquitination-dependent proteasomal degradation of p27 is well documented and shown to be regulated by different pathways (46, 82, 83). In general, although proteasomal degradation of short-lived proteins regulates cellular function of the protein as well as maintaining amino acid levels under normal conditions, during an extended period of nutrient starvation de novo synthesis and proteasomal degradation do not occur because of the lack of substrates and energy, and the vital amino acids are produced through autophagy (84). Our data demonstrate that either LC3 gene deletion or inhibition of autophagy with bafilomycin A1 showed no significant reduction of p27 level by TGF-β1 stimulation under the same serum deprivation conditions (Fig. 7). Moreover, blocking the ubiquitination-dependent proteasomal degradation by treatment with MG132 did not alter the p27 levels with or without TGF-β1 stimulation. These findings indicate that p27 is degraded through autophagy-mediated lysosomal degradation rather than ubiquitination-dependent proteasomal degradation pathway under serum deprivation.

In summary, we demonstrate that TGF-β1-induced autophagy protects glomerular mesangial cells from undergoing apoptosis under serum deprivation conditions through activation of the PI3K-Akt-mTOR-S6K signaling axis (Fig. 8). This is the first evidence that TGF-β1-induced Akt activation up-regulates autophagy and enhances cell cycle progression under serum deprivation conditions through regulating the protein levels of cyclins and cyclin-dependent kinase inhibitor p27 to promote mesangial cell survival.