HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Erratum All Versions of this Article: 19/7/837 04-2657fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wang, Q. Articles by McCulloch, C. A. Search for Related Content PubMed PubMed Citation Articles by Wang, Q. Articles by McCulloch, C. A.

Mitochondrial function is a critical determinant of IL-1-induced ERK activation

Qin Wang^*, Gregory P. Downey, Elena Bajenova^*, Maite Abreu, András Kapus and Christopher A. McCulloch^*,1

^* CIHR Group in Matrix Dynamics, University of Toronto, Toronto, Ontario M5S 3E2, Canada;
Division of Respirology, Department of Medicine, University of Toronto and the Research Institute, Toronto General Division of the University Health Network Research Institute, Toronto, Ontario, Canada; and
Department of Surgery, University of Toronto and the Division of Surgery, Toronto General Hospital, Toronto, Ontario, Canada

¹ Correspondence: Room 244, Fitzgerald Building, 150 College St., University of Toronto, Toronto, Ontario, Canada M5S 3E2. E-mail: christopher.mcculloch{at}utoronto.ca

SPECIFIC AIM

Compromised cellular energetics in inflammatory lesions can deregulate critical cell signaling pathways and perturb appropriate inflammatory responses. The aim of this study was to determine the impact of mitochondrial function on the regulation of interleukin 1 (IL-1) -induced ERK activation in human fibroblasts.

PRINCIPAL FINDINGS

1. Inhibition of cellular energetics by selective depolarization of mitochondria almost completely abolishes IL-1-induced cytosolic Ca²⁺ signals and ERK activation
We used cultured human gingival fibroblasts to study IL-1 signaling. These cells are important target cells for IL-1 in inflammatory lesions of the periodontium. In cells loaded with fura-2, IL-1 induced a high-amplitude, prolonged increase of calcium that was reduced slowly over time (Fig. 1 A). Dissipation of mitochondrial function and energetics by pretreatment with antimycin A and oligomycin or by depolarization with FCCP caused 3-fold reductions of ATP content and blocked IL-1-induced calcium increases.

View larger version (23K): [in this window] [in a new window]   Figure 1. Mitochondria affect IL-1-induced ERK activation. A) Cells in calcium buffer were pretreated with antimycin A + oligomycin or FCCP for 20 min, then incubated with IL-1. [Ca2+]i was measured in fura-2-loaded cells. Insets: Single cells pretreated with antimycin/oligomycin or FCCP, then exposed to ionomycin to establish cell viability and maintenance of intact calcium stores. B) Cells plated on fibronectin (FN) or poly-L-lysine (PL; to block IL-1-induced calcium release) were treated with IL-1. Phospho-ERK activity was assessed by immunoblotting for phospho-ERK. Total ERK was assessed by stripping and reprobing blots for ERK. C) Cells were untreated or preincubated with antimycin A + oligomycin FCCP for 20 min before addition of IL-1. D) Restoration of ERK activation after previous mitochondrial depolarization with antimycin A/oligomycin or FCCP was obtained by simultaneous addition of ionomycin and IL-1 for 15 min. E) Cells were pretreated with FCCP or with vehicle, then incubated with buffer (C or C+) or IL-1. Cells were probed for phospho-JNK and ß-actin for equal protein loading. Mitochondrial depolarization with FCCP does not block IL-1-induced JNK activation.

IL-1 induction of inflammatory mediators in many cases requires a priori activation of the MAP kinase ERK. Cells treated with IL-1 showed rapid induction of ERK activation (Fig. 1B ). If calcium release from internal stores was blocked by plating cells on poly-L-lysine before IL-1 stimulation, IL-1-induced ERK activation was blocked. When mitochondrial function and energetics were dissipated, IL-1-induced ERK activation was almost completely blocked (Fig. 1C ). This block could be overcome by treatment with ionomycin (Fig. 1D ), indicating that calcium is a key mediator in IL-1-induced ERK activation. The FCCP effect is specific to ERK and not to JNK (Fig. 1E ). Thus, mitochondrial function is critical for IL-1 signaling to ERK presumably because of its effect on calcium regulation.

2. IL-1-induced Ca²⁺ release from the endoplasmic reticulum requires mitochondrial Ca²⁺ uptake
We measured release of calcium from endoplasmic reticulum (ER) stores with the calcium indicator mag-fura-2, a dye that facilitates monitoring of [Ca²⁺]_ER. IL-1 induced rapid reduction of the mag-fura 2 ratio, followed by a swift recovery to baseline in cells plated on fibronectin (Fig. 2 A). There was no change of the mag-fura 2 ratio in IL-1-treated cells that had been preincubated with antimycin A and oligomycin or FCCP. Thus, mitochondrial function and energetics are required for IL-1-induced calcium release from ER stores.

View larger version (21K): [in this window] [in a new window]   Figure 2. Mitochondria are required for efficient Ca2+ release from ER. A) Measurement of [Ca2+]ER by mag-fura-2 ratio fluorescence. IL-1 induces rapid loss of the mag-fura-2 ratio, which is completely blocked by mitochondrial depolarization using antimycin/oligomycin or FCCP treatments. B) Whole cell calcium measurements [Ca2+]i in fura-2-loaded cells obtained in calcium-free medium. Carbachol or PGE2 treatments promote calcium release from intracellular stores; this release is reduced >5-fold by mitochondrial depolarization with antimycin/oligomycin. C) [Ca2+]i measurements in fura-2-loaded cells in calcium-free medium show sharp increases after thapsigargin (TG) treatments and little subsequent increase after ionomycin. Mitochondrial depolarization by FCCP (5 µM, 20 min) largely blocks thapsigargin-induced calcium increase (left panel) but ionomycin causes a prolonged increase of [Ca2+]i (right panel). D) Similar experimental design as panel C but cells were loaded with mag-fura-2 for estimates of [Ca2+]ER. In control cells, thapsigargin induces a sharp and rapid decrease of the mag-fura-2 ratio, which is not affected later by ionomycin. In contrast, after mitochondrial depolarization with FCCP, thapsigargin causes only a small and prolonged decrease of the mag-fura-2 ratio, which is strongly enhanced by subsequent treatment with ionomycin. E) Representative [Ca2+]mito traces of rhod-2-loaded cells plated on fibronectin pretreated with antimycin A + oligomycin (Anti. A/Oligo-pretreatment) or FCCP (FCCP-pretreatment), then treated with thapsigargin (1 µM). Each trace shows means ± SE of 4 or 5 independent samples. Aggregate data in histogram are from n = 4–5 experiments. Mitochondrial depolarization significantly inhibited IL-1-induced [Ca2+]m increases above baseline levels (P<0.001).

We determined whether the dramatic effect of mitochondrial inhibition on Ca²⁺ release from the ER was specific for IL-1. We measured [Ca²⁺]_i after stimulating control cells in Ca²⁺-free buffer with carbachol or PGE₂. In control cells, carbachol or PGE₂ treatments evoked a large transient indicative of Ca²⁺ release from internal stores. However, if mitochondria were depolarized by pretreatment with antimycin A + oligomycin (Fig. 2B ), the agonist-induced increase of [Ca²⁺]_i was reduced by 3-fold. Thus, in human gingival fibroblasts mitochondria are required for Ca²⁺ release from InsP₃-sensitive stores.

We assessed whether the agonist-induced efflux was suppressed or if mitochondria are essential for allowing Ca²⁺ efflux from the ER through the passive leak pathway. Thapsigargin induced a large transient increase of [Ca²⁺]_i above basal levels (Fig. 2C , left panel). In the absence of external Ca²⁺, if ionomycin was added after the thapsigargin-induced transient there were only marginal elevations of [Ca²⁺]_i, suggesting that ER stores had indeed been emptied by thapsigargin. In sharp contrast, if cells were pretreated with FCCP, thapsigargin induced only a very small increase of [Ca²⁺]_i; subsequent treatment with ionomycin provoked a well-defined rise (Fig. 2C , right panel). Thus, in the absence of mitochondrial function, even thapsigargin-induced Ca²⁺ release from the ER was impaired. To substantiate this, we monitored [Ca²⁺]_ER using ratio fluorimetry of mag-fura-2-loaded cells. In cells with functional mitochondria, thapsigargin induced rapid, large-amplitude reductions in the mag-fura-2 ratio. There was almost no response after subsequent treatment with ionomycin, indicating that ER calcium stores were depleted (Fig. 2D , left panel). If, on the other hand, cells were pretreated with FCCP, then thapsigargin was unable to reduce [Ca²⁺]_ER substantially while subsequent treatment with ionomycin reduced [Ca²⁺]_ER (Fig. 2D , right panel). These data show that independent of the type of stimulus, mitochondria are crucial for Ca²⁺ release from ER stores in human gingival fibroblasts.

3. Mitochondrial Ca²⁺ uptake and IL-1-induced Ca²⁺ release are coupled
We verified the functional coupling between ER stores and mitochondria by investigating the relationship between Ca²⁺ release from the ER and mitochondrial Ca²⁺ uptake. In control cells, thapsigargin induced a 4-fold increase of [Ca²⁺]_mito above basal levels (Fig. 2E , left panel; P <0.01, n=4). Pretreatment of cells with antimycin A/oligomycin or FCCP abrogated thapsigargin-induced [Ca²⁺]_mito transients (Fig. 2E , middle panels; n=4), as expected, indicating that Ca²⁺ release from the ER is coupled to mitochondrial Ca²⁺ uptake.

CONCLUSIONS

The novel findings of this study are 1) Inhibition of cellular energetics by selective depolarization of mitochondria strongly inhibits IL-1-induced cytosolic Ca²⁺ signals and ERK activation while preserving other important cellular functions; 2) IL-1-induced Ca²⁺ release from the endoplasmic reticulum requires mitochondrial Ca²⁺ uptake; 3) mitochondrial Ca²⁺ uptake and IL-1-induced Ca²⁺ release are coupled. To our knowledge, this is the first report of a specific mechanism by which depressed mitochondrial function, as seen in inflamed sites or in sepsis, leads to ineffective calcium regulation and subsequent block of MAP kinase activation in response to an inflammatory cytokine. As IL-1-induced activation of ERK is a critical signaling pathway for the expression and activation of matrix metalloproteinases in inflammation, our findings provide a functional link between cellular energetics and the dysregulation of matrix remodeling that occurs in infected sites and sepsis. The organization of the calcium signaling pathway and its reliance on mitochondrial function provide a specific functional explanation of how the shaping of calcium concentrations adjacent to the ER not only affects gating of store-operated channels, but activation of ERK (Fig. 3 ).

View larger version (26K): [in this window] [in a new window]   Figure 3. Mitochondrial regulation of calcium handling affects IL-1 induced ERK activation. The organization of the calcium signaling system affects the ability of IL-1 to activate ERK. After binding of IL-1 to the type 1 IL-1 signaling receptor, generation of an IP3-dependent signal stimulates calcium release from ER stores, a process tightly regulated by the ability of mitochondria to shape calcium signals. If mitochondrial energetics are dissipated and physiological calcium regulatory function of mitochondria is lost, calcium release from the ER in response to IL-1 is inhibited and ERK is not activated. The model provides a functional context by which mitochondrial energetics in inflammatory sites or sepsis plays a central regulatory role in mediating responses to inflammatory mediators such as IL-1.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2657fje;

This Article Full Text (PDF) Erratum All Versions of this Article: 19/7/837 04-2657fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wang, Q. Articles by McCulloch, C. A. Search for Related Content PubMed PubMed Citation Articles by Wang, Q. Articles by McCulloch, C. A.