Hypertonicity-enhanced TNF-α release from activated human monocytic THP-1 cells requires ERK activation Yung-Chen Chou a , Joen-Rong Sheu a , Chi-Li Chung b , Che-Jen Hsiao b , Po-Jen Hsueh a,c , George Hsiao a, ⁎ a Department of Pharmacology, Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan b Department of Chest Medicine, Taipei Medical University Hospital and School of Respiratory Therapy, Taipei Medical University, Taipei, Taiwan c Department of Otolaryngology, Head and Neck Surgery, Taipei Medical University Hospital, Taipei, Taiwan abstract article info Article history: Received 12 October 2010 Received in revised form 23 December 2010 Accepted 14 January 2011 Available online 20 January 2011 Keywords: Hypertonicity TNF-α LPS PMA Endocytosis TACE Background: Hypertonic stress enhances tumor necrosis factor (TNF)-α expression in activated monocytes. However, the underlying mechanism is unknown. The produced TNF-α is primarily cleaved and released by TNF-α-converting enzyme (TACE), and the surface expression of TACE is down-regulated by endocytosis. As hypertonicity inhibits endocytosis, we evaluated the mechanism of hypertonicity-induced TNF-α release from activated human monocytic THP-1 cells. Methods: THP-1 cells were stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) in the presence or absence of hypertonic agents (150 mM sucrose or 150–300 mM NaCl). The amount of TNF-α mRNA and protein, surface expression of TACE and activation of signaling pathways (mitogen- activated protein kinase, Akt and NF-κB) were assayed. Results: Hypertonic sucrose and NaCl significantly enhanced TNF-α release from THP-1 cells upon LPS or PMA stimulation. Hypertonic sucrose and other endocytosis inhibitors increased surface expression of TACE, but their effects on TNF-α release were inconsistent. This enhancement effect by hypertonicity was not attenuated by inhibition of TACE or IκB kinase, but it was blocked by cycloheximide and a MAP/ERK kinase inhibitor. The LPS- or PMA-induced TNF-α mRNA expression was not increased; rather, it was inhibited by hypertonicity. ERK1/2 was re-activated after sucrose treatment in LPS-stimulated THP-1 cells. Conclusions: Hypertonicity-enhanced TNF-α protein synthesis from LPS- or PMA-activated THP-1 cells requires ERK activation and may proceed without TACE. General significance: A vast amount of TNF-α production was regulated by a crucial post-transcriptional manner in activated human monocytic leukemia cells, and it may possibly be contributed to the cachexia condition. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Hyperosmolar status due to hyperglycemia is one of the most serious acute complications of diabetes mellitus [1]. In addition, patients with chronic renal failure who undergo intensive ultrafiltra- tion by peritoneal dialysis present higher levels of cytokines from hypertonicity [2,3]. Nevertheless, hypertonic solutions have been investigated for use as alternative resuscitation therapy for critically injured patients [4–6]. In cells, hypertonicity causes cytoskeletal rearrangement, inhibits mRNA transcription and protein translation, and depolarizes mitochondria [7]. However, hyperosmotic stress is considered to act as a stimulant and enhancer of proinflammatory cytokine expression [8,9]. The mechanism of hypertonicity-induced synergism on cytokine production has not yet been elucidated. Tumor necrosis factor (TNF)-α is a proinflammatory cytokine implicated in a variety of human diseases [10,11]. It is primarily produced by macrophages and monocytes in response to bacterial challenge or tumor-derived factors [12,13]. Additionally, TNF-α is a key mediator of fever and cachexia [14,15]. Overproduction of TNF-α is one indicator of acute symptoms of septic shock and the chronic effects of inflammatory diseases [16,17]. Monocytic cells can be induced to secrete TNF-α by two different pathways, the lipopoly- saccharide (LPS)-dependent and phorbol ester-dependent pathways [18]. LPS and phorbol 12-myristate 13-acetate (PMA) stimulate monocytes to produce TNF-α through activation of toll-like receptors and by stimulating protein kinase C (PKC), respectively [19,20]. Both of these events are involved in the nuclear factor (NF)-κB signaling pathway in response to TNF-α gene expression [21–23]. However, mitogen-activated protein kinases (MAPKs) such as p38 and ERK are signaling effectors that play important roles in the cellular response to Biochimica et Biophysica Acta 1810 (2011) 475–484 ⁎ Corresponding author at: Department of Pharmacology, Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110-31, Taiwan. Tel./fax: +886 2 27374622. E-mail address: geohsiao@tmu.edu.tw (G. Hsiao). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.01.004 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen