BackgroundHypoxia-mediated chemoresistance has been regarded as an important obstacle in the development of cancer treatment. Knockdown of krüppel-like factor 5 (KLF5) was reported to inhibit hypoxia-induced cell survival and promote cell apoptosis in non-small cell lung cancer (NSCLC) cells via direct regulation of hypoxia inducible factor-1α (HIF-1α) expression. However, the roles of KLF5 in the development of hypoxia-induced cisplatin (DDP) resistance and its underlying mechanism in NSCLC cells remain to be further elucidated.MethodsWestern blot was performed to determine the protein levels of KLF5, P-glycoprotein (P-gp) and HIF-1α in treated NSCLC cells. Cell survival was examined by MTT assay. The effect of KLF5 knockdown on hypoxia-induced glycolysis was assessed by measuring glucose consumption and lactate production. The effect of KLF5 knockdown on the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway was analyzed by western blot.ResultsHypoxia upregulated the expression of KLF5 in NSCLC cells. KLF5 knockdown suppressed hypoxia-induced DDP resistance in NSCLC cells, as demonstrated by the increased cytotoxic effects of DDP and reduced P-gp expression in NSCLC cells in hypoxia. Moreover, KLF5 knockdown inhibited hypoxia-induced HIF-1α expression and glycolysis, and KLF5 knockdown suppressed hypoxia-induced DDP resistance by inhibiting HIF-1α-dependent glycolysis in NSCLC cells. Furthermore, KLF5 knockdown suppressed hypoxia-induced activation of the PI3K/Akt/mTOR pathway in NSCLC cells and KLF5 overexpression promoted hypoxia-induced DDP resistance in NSCLC cells through activation of the PI3K/Akt/mTOR pathway.ConclusionsKLF5 knockdown could suppress hypoxia-induced DDP resistance, and its mechanism may be due to the inhibition of HIF-1α-dependent glycolysis via inactivation of the PI3K/Akt/mTOR pathway.
Organic anion transporter-3 (OAT3) is a member of the organic anion transporter family that mediates the body disposition of a diverse array of clinically important drugs. We previously demonstrated that activation of protein kinase C (PKC) inhibits OAT3 transport activity by accelerating OAT3 internalization from cell surface into intracellular compartments. In the current study, we established that PKC-induced inhibition of OAT3 transport activity in monkey kidney COS-7 cells and in human kidney HEK293 cells occurred through an enhanced OAT3 ubiquitination, a process catalyzed by an E3 ubiquitin-protein ligase Nedd4-2 (neural precursor cell expressed, developmentally down-regulated 4-2). Overexpression of Nedd4-2 enhanced OAT3 ubiquitination, decreased OAT3 expression at the cell surface, and inhibited OAT3 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-2/C821A or siRNA knockdown of endogenous Nedd4-2 had opposite effects on OAT3. Furthermore, immunoprecipitation experiments conducted both in culture cells and with rat kidney slices showed that there was a physical interaction between OAT3 and Nedd4-2. In conclusion, our results provided the first evidence that Nedd4-2 is an important regulator for OAT3 ubiquitination, expression and transport activity.
Human organic anion transporter 1 (hOAT1) expressed at the membrane of the kidney proximal tubule cells mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and antiinflammatories. Therefore, understanding the regulation of hOAT1 will provide significant insights into kidney function and dysfunction. We previously established that hOAT1 transport activity is inhibited by activation of protein kinase C (PKC) through accelerating hOAT1 internalization from cell surface into intracellular endosomes and subsequent degradation. We further established that PKC-induced hOAT1 ubiquitination is an important step preceding hOAT1 internalization. In the current study, we identified two closely related E3 ubiquitin ligases, neural precursor cell expressed, developmentally downregulated 4-1 and 4-2 (Nedd4-1 and Nedd4-2), as important regulators for hOAT1: overexpression of Nedd4-1 or Nedd4-2 enhanced hOAT1 ubiquitination, reduced the hOAT1 amount at the cell surface, and suppressed hOAT1 transport activity. In further exploring the relationship among PKC, Nedd4-1, and Nedd4-2, we discovered that PKC-dependent changes in hOAT1 ubiquitination, expression, and transport activity were significantly blocked in cells transfected with the ligase-dead mutant of Nedd4-2 (Nedd4-2/C821A) or with Nedd4-2-specific siRNA to knockdown endogenous Nedd4-2 but not in cells transfected with the ligase-dead mutant of Nedd4-1 (Nedd4-1/C867S) or with Nedd4-1-specific siRNA to knockdown endogenous Nedd4-1. In conclusion, this is the first demonstration that both Nedd4-1 and Nedd4-2 are important regulators for hOAT1 ubiquitination, expression, and function. Yet they play distinct roles, as Nedd4-2 but not Nedd4-1 is a critical mediator for PKC-regulated hOAT1 ubiquitination, expression, and transport activity.
Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT4 is abundantly expressed in the kidney and placenta. In the current study, we examined the regulation of hOAT4 by serum- and glucocorticoid-inducible kinase 2 (sgk2) in the kidney COS-7 cells. We showed that sgk2 stimulated hOAT4 transport activity. Such stimulation mainly resulted from an increased cell surface expression of the transporter, kinetically revealed as an increased maximal transport velocity Vmax without significant change in substrate-binding affinity Km. We further showed that regulation of hOAT4 activity by sgk2 was mediated by ubiquitin ligase Nedd4-2. Overexpression of Nedd4-2 enhanced hOAT4 ubiquitination, and inhibited hOAT4 transport activity, whereas overexpression of ubiquitin ligase-dead mutant Nedd4-2/C821A or siRNA knockdown of endogenous Nedd4-2 had opposite effects on hOAT4. Our co-immunoprecipitation experiment revealed that sgk2 weakened the association between hOAT4 and Nedd4-2. In conclusion, our study demonstrated for the first time that sgk2 stimulated hOAT4 transport activity by abrogating the inhibition effect of Nedd4-2 on the transporter.
Synopsis Organic anion transporters (OATs) encoded by solute carrier 22 (SLC22) family are localized in the epithelia of multiple organs, where they mediate the absorption, distribution, and excretion of a diverse array of negatively charged environmental toxins and clinically important drugs. Alterations in the expression and function of OATs play important roles in intra- and inter-individual variability of the therapeutic efficacy and the toxicity of many drugs. As a result, the activity of OATs must be under tight regulation so as to carry out their normal functions. The regulation of OAT transport activity in response to various stimuli can occur at several levels such as transcription, translation, and posttranslational modification. Posttranslational regulation is of particular interest, because it usually happens within a very short period of time (minutes to hours) when the body has to deal with rapidly changing amounts of substances as a consequence of variable intake of drugs, fluids, or meals as well as metabolic activity. This review article highlights the recent advances from our laboratory in uncovering several posttranslational mechanisms underlying OAT regulation. These advances offer the promise of identifying targets for novel strategies that will maximize therapeutic efficacy in drug development.
Organic anion transporter 3 (OAT3) plays a vital role in removing a broad variety of anionic drugs from kidney, thus avoiding their possible toxicity in the body. We earlier established that activation of protein kinase C (PKC) enhances OAT3 ubiquitination, which promotes OAT3 internalization from the cell plasma membrane to intracellular endosomes and consequent degradation. As a result, OAT3 expression and transport activity are reduced. In the current study, we discovered that protein kinase A (PKA) had an opposite effect to PKC on the regulation of OAT3. We showed that activation of PKA by Bt2-cAMP stimulated OAT3 transport activity, which was largely caused by an enhanced plasma membrane expression of the transporter, kinetically reflected as an augmented maximal transport velocity Vmax without notable alteration in substrate-binding affinity Km. Additionally, we showed that PKA activation accelerated the rate of OAT3 recycling from intracellular compartments to the plasma membrane and decelerated the rate of OAT3 degradation. We further showed that OAT3 is subjected to post-translational modification by SUMO-2 and SUMO-3 not by SUMO-1. PKA activation enhanced OAT3 SUMOylation, which was accompanied by a reduced OAT3 ubiquitination. Finally, insulin like growth factor 1 significantly stimulated OAT3 transport activity and SUMOylation through PKA signaling pathway. In conclusion, this is the first demonstration that PKA stimulated OAT3 expression and transport activity by altering the trafficking kinetics of OAT3 possibly through the crosstalk between SUMOylation and ubiquitination.
Human organic anion transporter 1 (hOAT1), expressed at the basolateral membrane of kidney proximal tubule cells, mediates the active renal secretion of a diverse array of clinically important drugs, including anti-human immunodeficiency virus therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We have previously demonstrated that posttranslational modification of hOAT1 by ubiquitination is an important mechanism for the regulation of this transporter. The present study aimed at identifying the ubiquitin ligase for hOAT1 and its mechanism of action. We showed that overexpression of neural precursor cell expressed, developmentally downregulated (Nedd)4-1, an E3 ubiquitin ligase, enhanced hOAT1 ubiquitination, decreased hOAT1 expression at the cell surface, and inhibited hOAT1 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-1/C867S was without effects on hOAT1. Furthermore, knockdown of endogenously expressed Nedd4-1 by Nedd4-1-specific small interfering RNA reduced hOAT1 ubiquitination. Immunoprecipitation experiments in cultured cells and rat kidney slices and immunofluorescence experiments in rat kidney slices showed that there was a physical interaction between OAT1 and Nedd4-1. Nedd4-1 contains four protein-protein interacting WW domains. When these WW domains were inactivated by mutating two amino acid residues in each of the four WW domains (Mut-WW1: V210W/H212G, Mut-WW2: V367W/H369G, Mut-WW3: I440W/H442G, and Mut-WW4: I492W/H494G, respectively), only Mut-WW2 and Mut-WW3 significantly lost their ability to bind and to ubiquitinate hOAT1. As a result, Mut-WW2 and Mut-WW3 were unable to suppress hOAT1-mediated transport as effectively as wild-type Nedd4-1. In conclusion, this is the first demonstration that Nedd4-1 regulates hOAT1 ubiquitination, expression, and transport activity through its WW2 and WW3 domains.
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