Involvement of S6K1 in mitochondria function and structure in HeLa cells☆
Introduction
Mitochondria are an important source of energy for cells. Therefore, they represent an important intracellular organelle in most cell types. Mitochondria also play a key role in apoptosis initiation, and are involved in a range of pathologic conditions including cancer, neurodegeneration, aging, and inflammation [1]. Following Warburg's theory of metabolic alteration in response to glycolytic production of ATP, despite the presence of oxygen in tumor cells, mtDNA mutations and mitochondrial dysfunctions have been widely investigated to explore the mechanisms underlying malfunction of the respiratory chain, which forces cells to increase glycolysis to maintain their ATP supply [2]. The conversion of glucose to lactate, which can occur in normal cells under hypoxic conditions, still occurs in cancer tissues despite the presence of oxygen that would normally prevent glycolysis through the Pasteur effect. It is now clear that persistent aerobic glycolysis in certain cancer cells is associated with the activation of oncogenes or inactivation of tumor suppressors [3], [4].
Mitochondria function as essential regulators of cell death, including apoptotic and necrotic cell death. Therefore, the control of mitochondria must be tightly regulated to prevent cell death [5]. In addition, mitophagy is considered as a key process of quality control, which is a selective part of autophagy to remove damaged or unnecessary mitochondria [6]. Mitochondria are dynamic organelles that always experience fission and fusion. This mitochondrial dynamics are required for cell adaptation to alter the conditions desired for cell growth, division, and distribution of mitochondria during differentiation [7]. Mitochondrial fusion in mammals is mediated by the fusion proteins mitofusin 1 (Mfn1), Mfn2, and optic atrophy 1 (OPA1). Mfn1 and Mfn2 are dynamin-related GTPases required for the fusion of outer mitochondrial membranes. OPA1 is also a dynamin-related GTPase, required for fusion of the inner mitochondrial membranes [8]. Mitochondrial fission is mediated by dynamin-related protein 1 (Drp1), which is a large GTPase. Drp1 is a cytosolic protein that can be translocated to the outer mitochondrial membrane, leading to mitochondrial fission. Drp1 interacts with four mitochondrial receptor proteins: fission 1 (Fis1), mitochondria fission factor (Mff), and mitochondrial dynamics protein of 49 kDa and 51 kDa (MiD49 and MiD51, respectively) [9], [10], [11].
Ribosomal protein S6 kinase beta-1 (S6K1, also known as p70S6 kinase-p70S6K1) is a protein kinase that can phosphorylate S6, leading to protein synthesis at the ribosome [12]. S6K1 acts downstream of PI3K signaling pathways [13]. It has been shown that S6K1-deficient mice are resistant to obesity due to the enhancement of beta-oxidation [14]. Inhibition or deletion of S6K1 also resulted in delayed production of fat cells by blocking the initial stage of adipocyte formation. This indicates that the function of S6K1 in adipocytes is associated with obesity [15]. S6K1 is also involved in muscle lengthening and growth [16]. Inactive S6K1 can bind to eukaryotic initiation factor 3 (eIF3) and is eliminated by mTOR/Raptor-mediated phosphorylation. The resulting free S6K1 can phosphorylate its downstream targets, including S6 [17], [18].
In the current study, we found that lentivirus-mediated S6K1 knockdown resulted in fragmented mitochondria with enhanced oxygen consumption. In addition, S6K1 depletion leads to the enhancement of ATP production in cytoplasmic and mitochondrial ATP. However, the relative ratio of mitochondria-dependent ATP production is decreased in lenti-shS6K1-HeLa cells compared to control cells. Taken together, our results clearly suggest that S6K1 may be involved in the regulation of mitochondria morphology and function in HeLa cells.
Section snippets
Antibodies and reagents
All commercial antibodies were purchased from the following: Anti-S6K1 antibody (Abnova); Anti-Mfn2, anti-Opa1, anti-Drp1 and anti-Fis1 (Abcam); Anti-VDAC, anti-AIF, anti-pT389-S6K1 and anti-COX4 (Cell signaling); anti-Hsp60 and anti-Actin antibodies (Sigma-Aldrich). Horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibodies were purchased from Invitrogen. CCCP was purchased from Sigma-Aldrich. Rapamycin and staurosporin were purchased from Calbiochem.
Cell culture and stimulation
HeLa cells
Reduction of S6K1 in HeLa cells results in mitochondrial fission
Based on studies of S6K1 function in early adipocyte differentiation [15], stable HeLa cells for S6K1 knockdown were generated using shS6K1-lentivirus. We observed morphological changes in mitochondria in S6K1-knowdowned HeLa cells (Fig. 1A) based on co-expression of Mito Red. Therefore, detailed image analysis for S6K1-mediated mitochondria shape was performed in shS6K1-HeLa stable cells using several different methods. Elongation shape factor is defined as the square root of the ratio of the
Discussion
S6 kinase 1 (S6K1) modulates nutrient and growth factor signals to control cell growth and survival [29]. S6K1 also plays a role in regulating energy metabolism in vivo, since S6K1-knockout mice are resistant to obesity due to enhanced beta-oxidation with an increased number of large mitochondria [14]. Unexpectedly, it has been found in studies of S6K1 function in early adipocyte differentiation that S6K1 depletion results in mitochondrial fragmentation in lenti-shS6K1-HeLa cells (Fig. 1, Fig. 2
Conflict-of-interest/financial disclosure statement
None.
Acknowledgement
We would like to thank Dr. David Plas (Univ. of Cincinnati) for providing shS6K1-FL5.12 stable cells. This work was financially supported by National Research Foundation of Korea (NRF) grants from the Korean Government (MEST) (NRF-2012M3A9B6055302, NRF-2014R1A1A3050752, NRF-2015R1A2A2A01003597, NRF-2015R1D1A3A0101569 4).
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