The protective role of autophagy in experimental osteoarthritis, and the therapeutic effects of Torin 1 on osteoarthritis by activating autophagy

Cheng, Nitao; Ai, Guo; Meng, Hui

doi:10.1186/s12891-016-0995-x

Cited by 45 publications

(53 citation statements)

References 50 publications

(53 reference statements)

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“…Torin 1 [84] and pp242 [85] have been reported to induce autophagy on account of their inhibition of MTOR activity. However, we found that Torin 1 and pp242 treatment did not enhance autophagy flux in HaCaT cells, although the increase in the conversion from LC3-I to LC3-II was observed in cells treated with them.…”

Section: Discussionmentioning

confidence: 99%

Impact on Autophagy and Ultraviolet B Induced Responses of Treatment with the MTOR Inhibitors Rapamycin, Everolimus, Torin 1, and pp242 in Human Keratinocytes

et al. 2017

Oxidative Medicine and Cellular Longevity

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The mechanistic target of Rapamycin (MTOR) protein is a crucial signaling regulator in mammalian cells that is extensively involved in cellular biology. The function of MTOR signaling in keratinocytes remains unclear. In this study, we detected the MTOR signaling and autophagy response in the human keratinocyte cell line HaCaT and human epidermal keratinocytes treated with MTOR inhibitors. Moreover, we detected the impact of MTOR inhibitors on keratinocytes exposed to the common carcinogenic stressors ultraviolet B (UVB) and UVA radiation. As a result, keratinocytes were sensitive to the MTOR inhibitors Rapamycin, everolimus, Torin 1, and pp242, but the regulation of MTOR downstream signaling was distinct. Next, autophagy induction only was observed in HaCaT cells treated with Rapamycin. Furthermore, we found that MTOR signaling was insensitive to UVB but sensitive to UVA radiation. UVB treatment also had no impact on the inhibition of MTOR signaling by MTOR inhibitors. Finally, MTOR inhibition by Rapamycin, everolimus, or pp242 did not affect the series of biological events in keratinocytes exposed to UVB, including the downregulation of BiP and PERK, activation of Histone H2A and JNK, and cleavage of caspase-3 and PARP. Our study demonstrated that MTOR inhibition in keratinocytes cannot always induce autophagy, and the MTOR pathway does not play a central role in the UVB triggered cellular response.

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Section: Discussionmentioning

confidence: 99%

Impact on Autophagy and Ultraviolet B Induced Responses of Treatment with the MTOR Inhibitors Rapamycin, Everolimus, Torin 1, and pp242 in Human Keratinocytes

et al. 2017

Oxidative Medicine and Cellular Longevity

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“…In vitro , autophagic inhibition stimulates apoptosis and an increase of matrix metalloproteinases in chondrocytes [5, 6]. Specifically, the relationship between compromised autophagy and the severity and progression of OA has been confirmed in many studies [7, 8]. Thus, in the progression of cartilage degeneration, finding a cause for the inhibition of chondrocyte autophagy during OA may become a potential strategy against this disease.…”

Section: Introductionmentioning

confidence: 99%

Transient Receptor Potential Vanilloid 5 Mediates Ca2+ Influx and Inhibits Chondrocyte Autophagy in a Rat Osteoarthritis Model

Wei¹,

Wang²,

Wang³

et al. 2017

Cell Physiol Biochem

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Background: Autophagy, a self-protective mechanism of chondrocytes, has become a promising target to impede the progress of osteoarthritis (OA). Autophagy is regulated by cytosolic Ca²⁺ activity and may thus be modified by the Ca²⁺ permeable transient receptor potential channel vanilloid 5 (TRPV5). Therefore, we investigated the potential role of TRPV5 in mediating Ca²⁺ influx and in inhibiting chondrocyte autophagy in a rat OA model. Methods: The rat OA model was assessed by macroscopic and histological analyses. light chain 3B (LC3B) immunolocalization was detected by immunohistochemistry. TRPV5, LC3B and calmodulin in OA articular cartilage were assessed by real time polymerase chain reaction (RT-PCR) and western blotting. TRPV5 small interfering RNA (TRPV5 siRNA) were transfected into rat primary chondrocyte then the calmodulin and LC3B was detected by immunofluorescence. The functionality of the TRPV5 was assessed by Ca²⁺ influx. Western blot was used to measure autophagy-related proteins. Results: We constructed a monosodium iodoacetate (MIA) -induced rat OA model and found that ruthenium red (TRPV5 inhibitor) slowed the progression of joint destruction. We found that the TRPV5 and calmodulin were up-regulated but LC3B was down-regulated in articular cartilage following prolonged progression of OA. Furthermore, the up-regulated TRPV5 channel caused an increase in the Ca²⁺ influx in chondrocytes. The up-regulation of TRPV5 stimulated Ca²⁺ influx, which inhibited autophagy by increasing the production of calmodulin, phosphorylation of calmodulin dependent protein kinases II (p-CAMK II), phosphorylation of Beclin1 (p-Beclin1), and protein of B-cell lymphoma-2 (Bcl-2), and attenuating ratio of LC3-II/ LC3-. Conclusion: Up-regulated TRPV5 as an initiating factor inhibited chondrocyte autophagy via the mediation of Ca²⁺ influx.

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“…For inhibitor treatment in 2D and 3D cultures, 100 nM of rapamycin (Grabiner et al, 2014;Kang et al, 2013;Sarbassov et al, 2006), 250 nM of Torin1 (Cheng et al, 2016;Grabiner et al, 2014;Kang et al, 2013;Thoreen et al, 2012), 10 μM of PF47068671 (Pearce et al, 2010;Schipany et al, 2015;Zhang et al, 2015), 1 μM of MK2206 (Devery et al, 2015;Shen et al, 2015;Sung et al, 2016) and 1 μM of AZD6244 (Devery et al, 2015;Ewald et al, 2015) were employed as these concentrations have been most frequently used in cell-based assays and have a complete effect on their respective targets [ phospho-protein analysis in western blots (see Fig. 4 and western blots in the supporting figures)].…”

Section: Inhibitor Treatmentmentioning

confidence: 99%

Comparison of cancer cells cultured in 2D vs 3D reveals differences in AKT/mTOR/S6-kinase signaling and drug response

Riedl

Schlederer

Pudelko

et al. 2016

Journal of Cell Science

358

293

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Three-dimensional (3D) cancer models are used as preclinical systems to mimic physiologic drug responses. We provide evidence for strong changes of proliferation and metabolic capacity in three dimensions by systematically analyzing spheroids of colon cancer cell lines. Spheroids showed relative lower activities in the AKT, mammalian target of rapamycin (mTOR) and S6K (also known as RPS6KB1) signaling pathway compared to cells cultured in two dimensions. We identified spatial alterations in signaling, as the level of phosphorylated RPS6 decreased from the spheroid surface towards the center, which closely coordinated with the tumor areas around vessels in vivo. These 3D models displayed augmented antitumor responses to AKT-mTOR-S6K or mitogen-activated protein kinase (MAPK) pathway inhibition compared to those in 2D models. Inhibition of AKT-mTOR-S6K resulted in elevated ERK phosphorylation in 2D culture, whereas under these conditions, ERK signaling was reduced in spheroids. Inhibition of MEK1 (also known as MAP2K1) led to decreased AKT-mTOR-S6K signaling in 3D but not in 2D culture. These data indicate a distinct rewiring of signaling in 3D culture and during treatment. Detached tumor-cell clusters in vessels, in addition to circulating single tumor cells, play a putative role in metastasis in human cancers. Hence, the understanding of signaling in spheroids and the responses in the 3D models upon drug treatment might be beneficial for anti-cancer therapies.

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The protective role of autophagy in experimental osteoarthritis, and the therapeutic effects of Torin 1 on osteoarthritis by activating autophagy

Cited by 45 publications

References 50 publications

Impact on Autophagy and Ultraviolet B Induced Responses of Treatment with the MTOR Inhibitors Rapamycin, Everolimus, Torin 1, and pp242 in Human Keratinocytes

Impact on Autophagy and Ultraviolet B Induced Responses of Treatment with the MTOR Inhibitors Rapamycin, Everolimus, Torin 1, and pp242 in Human Keratinocytes

Transient Receptor Potential Vanilloid 5 Mediates Ca2+ Influx and Inhibits Chondrocyte Autophagy in a Rat Osteoarthritis Model

Comparison of cancer cells cultured in 2D vs 3D reveals differences in AKT/mTOR/S6-kinase signaling and drug response

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