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CELLULAR METABOLISM
1Department of Medicine, Section of Nephrology, and 2Department of Physiology and Biophysics, University of Illinois at Chicago College of Medicine; and 3Veterans Affairs Chicago Health Care System, Chicago, Illinois 60612
Submitted 2 April 2003 ; accepted in final form 6 April 2004
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ABSTRACT |
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increased HK activity in both a time- and concentration-dependent manner: activity increased maximally by 
50% between 12 and 24 h with an apparent EC50 of 3 pM. IL-1
mimicked, but did not augment, the effects of IL-1. Specific IL-1 receptor antagonism and selective MAPK/ERK kinase or upstream Ras inhibition prevented these increases, whereas PKC inhibition did not. Changes in HK activity were associated with both increased Glc metabolism and selective increases in HKII isoform abundance. We conclude that IL-1 cytokines can regulate cellular Glc phosphorylating capacity via an IL-1 receptor-, Ras-, and classic MAPK pathway-mediated increase in HKII abundance. These findings suggest a novel, previously undescribed mechanism whereby metabolism may be coupled to inflammation and injury. mitogen-activated protein kinase; glucose; metabolism; Ras; inflammation
Interleukin-1 cytokines (IL-1 and IL-1) are proinflammatory polypeptides with a broad range of biological activities (16). IL-1 of both glomerular (38, 65, 71, 72) and inflammatory cell (20, 37) origin has been implicated in the initiation and progression of glomerular injury (63, 65), as well as associated reparative events (43). These pleiotropic cytokines are also capable of activating the classic MAPK pathway in mesangial cells (31, 57, 67), which plays a central role in HK regulation in this cell type (14, 53–55). Direct cytokine regulation of HK activity, however, has not been previously reported, so we examined the ability of IL-1 to influence HK activity and expression in cultured murine mesangial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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/PKC pseudosubstrate sequence (myr-FARKGALRQ; myrPKC/20-28), the conserved C2 domain of conventional PKC (myr-SLNPEWNET; myrPKCC2-4), and the unique V1 region of PKC
(myr-EAVSLKPT; myrPKCV1-2) were obtained from Biomol (Plymouth Meeting, MA). Yeast Glc-6-P dehydrogenase (G6PDH) was obtained from Roche Molecular Biochemicals (Indianapolis, IN). The recombinant murine IL-1 receptor antagonist, IL-1ra, was obtained from R&D Systems (Minneapolis, MN). All cell culture reagents, including serum and additives, were purchased from Invitrogen (Grand Island, NY). All other reagents, including recombinant murine IL-1 and IL-1 cytokines, phorbol 12-myristate 13-acetate (PMA), NADP, and ATP, were obtained from Sigma (St. Louis, MO) unless otherwise noted. Cell culture. Mycoplasma-free SV40 MES 13 (murine mesangial) cells were obtained from American Type Culture Collection (Rockville, MD) at passage 27. These cells are derived from glomerular explants of SV40 transgenic mice and exhibit both biochemical and morphological features of normal mesangial cells in culture (39, 41, 53). Cell monolayers were routinely maintained in HEPES-buffered (14 mM) DME-F-12 (3:1) medium containing 6 mM Glc and supplemented with 5% fetal bovine serum. Cells were routinely grown to confluence in a humidified 37°C, 5% CO2 incubator before testing, and all experiments were performed between passages 30 and 40. Where appropriate, cells were serum-deprived for 16–24 h before and during testing. When inhibitors were employed, cells were typically pretreated with inhibitor alone for at least 0.5 h before testing.
Adenoviral gene transfer.
Forced transgene expression was accomplished using replication-deficient recombinant adenoviruses as described previously (10, 25). pACCMVpLpA-based vectors (5) expressing constitutively active (S217E/S221E) and dominant interfering (S221A) mutants of rabbit MEK1 have been described previously (34) and were the generous gift of Dr. Jeffery D. Molkentin (University of Cincinnati, Cincinnati, OH). Corresponding vectors expressing the dominant interfering K17N mutant form of human H-Ras and a prokaryotic -galactosidase reporter gene have also been described (5, 23) and were obtained from Drs. Barbara B. Kahn (Harvard University, Cambridge, MA) and Christopher B. Newgard (Duke University, Durham, NC), respectively. A series of pAdEasy-derived vectors (Stratagene, La Jolla, CA) expressing dominant interfering mutants of bovine PKC (K368R), rat PKC (K436R), and human PKC
(K409R), as well as a constitutively active rat PKC mutant (A159E), were obtained from Dr. Trevor J. Biden (Garvan Institute of Medical Research, Sydney, Australia) and were used as described previously (13).
Cell lysate preparation. Whole cell lysates suitable for both HK activity assays and immunoblot analysis were prepared by brief sonication (30–60 J) in ice-cold lysis buffer containing 45 mM Tris·HCl, 50 mM KH2PO4, 10 mM Glc, 11.1 mM monothioglycerol, 0.5 mM EDTA, and 0.2% (vol/vol) Triton X-100, pH 8.2. Consistent with a previous report (27) that Glc stabilizes HKs and decreases their proteolytic susceptibility in vitro, we found the routine use of protease inhibitors to be unnecessary in the analysis of both HK activity and isoform abundance in preparations in which the Glc content was maintained at 10 mM (data not shown). After centrifugation at 11,900 g and 4°C for 10 min to pellet insoluble debris, aliquots of the resulting supernatants were immediately assayed for total HK activity, and the remainder was stored at –30°C for subsequent immunoblot analysis.
HK assays.
HK activity was measured as the total Glc phosphorylating capacity of fresh whole cell lysates by using a standard G6PDH-coupled assay as described previously (54). In brief, the rate of Glc- and ATP-dependent NADP reduction by fresh cell lysates was assayed spectrophotometrically in the presence of nonlimiting G6PDH. The final assay mixture consisted of 1 U/ml G6PDH, 0.5 mg/ml NADP, 6.7 mM ATP, 7 mM MgCl2, 4 mM Glc, 2.5 mM KH2PO4, 1 mM NaH2PO4, 11.1 mM monothioglycerol, 0.01% (vol/vol) Triton X-100, 0.025 mM EDTA, and 45 mM Tris·HCl, pH 8.5. HK activity was routinely measured at 25°C under established linear assay conditions (54), and the corresponding total cellular protein content was determined according to the method of Bradford (9), using bovine 
-globulin (Bio-Rad) as a reference standard. Specific activities were calculated as units (U) per gram of protein, where 1 U is defined as the enzyme activity required for the coupled formation of 1 nmol of NADPH per minute at 25°C (millimolar extinction coefficient 6.22 at 340 nm). These activities are reported as percent activity relative to unstimulated time-paired controls to facilitate comparisons.
Extracellular signal-regulated kinase activation assays. Extracellular signal-regulated kinase (ERK) activation was assayed by a specific immunoprecipitated kinase (IP/kinase) activity assay as described previously (53, 55). In brief, activated ERK1/2 immunoprecipitates were prepared from cell lysates by using immobilized monoclonal antibodies directed against the dual-phosphorylated pTEpY activation motif of ERK1/2. Immunoprecipitates were then assayed for the ability to specifically serine phosphorylate an Elk-1 fusion protein in vitro. Total phosphotransferase activity was assessed by immunoblot analysis by using rabbit polyclonal IgG specific for phospho-Elk-1 (Elk-1-P) and a commercially available chemiluminescent detection system (Phototope-HRP; Cell Signaling Technology, Beverly, MA). Control IP/kinase assays were routinely performed in parallel by using unstimulated cell lysates with and without the addition of functional MEK-activated recombinant ERK2 (Cell Signaling Technology). Major results were confirmed by quantitative assessment of specific ERK1/2 phosphorylation as described previously (54). Densitometric analysis of individual autoradiograms was performed using digital images acquired by an Eagle-Eye II still video imaging system (Stratagene) and public domain NIH Image 1.63 software for Macintosh computers (National Institutes of Health, Bethesda, MD).
Glc utilization and lactate production assays.
Glc utilization and lactate production were assayed as net disappearance of Glc and net accumulation of lactate in the culture medium, respectively. Cells were routinely tested in serum-free growth medium containing 6 mM Glc and lacking the pH indicator phenol red, which interferes with these chromogenic assays. At appropriate time points, medium aliquots were assayed spectrophotometrically for both Glc and lactate content via standard enzymatic coupled reactions as described previously (53, 55). All measures of medium Glc and lactate content were performed in the presence of nonlimiting concentrations of Glc and under conditions of linear net Glc utilization and lactate accumulation.
Immunoblot analysis. Whole cell lysates were electrophoretically resolved and transferred to nitrocellulose membranes for immunoblotting as described previously (53, 55). Blots were routinely stained with 0.1% (wt/vol) Ponceau S in 5% (vol/vol) acetic acid to confirm both the uniformity of gel loading and the efficiency of membrane transfer. To minimize nonspecific binding, blots were routinely washed with Tween 20-containing Tris-buffered saline [TTBS; 100 mM Tris·HCl, 0.9% (wt/vol) NaCl, and 0.1% (vol/vol) Tween 20, pH 7.5] and preincubated in TTBS supplemented with 5% (wt/vol) nonfat dry milk for 1 h at 25°C. Blots were then incubated with primary antibodies in TTBS containing 5% BSA overnight at 4°C or for 4 h at 25°C before probing with matched secondary antibodies in TTBS containing 5% nonfat milk for 1 h at 25°C. Specific rabbit polyclonal antipeptide antisera directed against the carboxy-terminal 18 residues of rat/human HKII were used for all HKII immunoblots and were either generated commercially (ResGen, Huntsville, AL) or generously provided by Dr. Daryl K. Granner (Vanderbilt University, Nashville, TN). Rabbit polyclonal antipeptide antisera directed against the carboxy-terminal 11 residues of human HKI and against the carboxy-terminal 9 residues of human HKIII were also generated commercially (Alpha Diagnostic International, San Antonio, TX). Rat brain lysates, recombinant human HKII (the gift of Richard L. Printz and Daryl K. Granner, Vanderbilt University), and mouse lung lysates were routinely used as positive controls in HKI, HKII, and HKIII immunoblot analysis, respectively. Specific protein bands were visualized using the Phototope-HRP chemiluminescent detection system (Cell Signaling Technology). Specificity was addressed by parallel analysis with the use of preimmune serum or immunoglobulin controls where appropriate. The ability of these antibody preparations to specifically identify their target isoforms was validated by the parallel use of independent isoform-specific antibody preparations obtained commercially (Santa Cruz Biotechnology or Chemicon) or kindly provided by Drs. Daryl K. Granner (Vanderbilt University) and John E. Wilson (Michigan State University, East Lansing, MI). Quantitative densitometric analysis was performed as detailed above.
Statistical analysis. All data are presented as means ± SE for at least three independent measurements unless otherwise noted. Statistical comparisons were performed using either two-tailed paired t-test or analysis of variance with Scheffé's F procedure for post hoc comparisons where appropriate, using a significance level of 95% and StatView 5.0.1 software for Macintosh computers (SAS Institute, Cary, NC).
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RESULTS |
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increases mesangial cell HK activity in a time- and concentration-dependent manner.
As shown in Fig. 1, 50 pM IL-1 increased total HK activity in cultured mesangial cells by >15% within 4 h (P = 0.08) and by nearly 40% at 8 h (P < 0.01). HK activity was also maximally increased by nearly 60% (P < 0.001) between 12 and 24 h after IL-1 exposure. As depicted in Fig. 2, this stimulatory effect of IL-1 also exhibited concentration dependence. Total HK activity was significantly increased by 
1 pM IL-1 at 24 h (P < 0.01). This effect was saturable, with maximal stimulation observed with 10 pM IL-1 and an apparent EC50 of 3 pM (51 pg/ml).
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and IL-1 are mediated by the type I IL-1 receptor (17), which is constitutively expressed by mesangial cells (71, 72). The saturable capacity of IL-1 to increase HK activity (Fig. 2) is kinetically compatible with type I IL-1 receptor activation. However, as an indirect test of this receptor's involvement in IL-1-stimulated HK activity, we also examined the ability of IL-1 to mimic the stimulatory effect of IL-1 on total HK activity in mesangial cells. As shown in Fig. 3, both IL-1 and IL-1, at a concentration of 50 pM, increased total HK activity by >50% at 24 h (P 
0.005). In combination, the effects of these cytokines were nonadditive, consistent with a common mechanism of action, presumably via the type I IL-1 receptor. To more directly address this issue, we also examined the specific naturally occurring IL-1 receptor antagonist, IL-1ra (17, 18, 21), for the ability to prevent IL-1 stimulation of HK activity. As shown in Fig. 4, IL-1ra prevented IL-1-stimulated HK activity in a concentration-dependent manner (an apparent IC50 of 89 ng/ml or 5 nM; P < 0.05). In contrast, IL-1ra had no corresponding effect on basal HK activity at the same concentrations, suggesting that this antagonist does not directly inhibit HK activity.
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is a known activator of the classic MAPK pathway in this cell type, we also examined the ability of both PD-98059 and U0126 to prevent HK induction by IL-1. As depicted in Fig. 5A, pretreatment with either antagonist inhibited the induction of HK activity by 50 pM IL-1 at 24 h. The inhibitory effects of both PD-98059 and U0126 were concentration dependent and were maximally effective at levels deemed specific for MEK. These concentrations also correspond to those shown previously to prevent both ERK activation and increased HK activity in this cell type (14). The ability of forced expression of a dominant interfering mutant of MEK1 to inhibit (Fig. 5B; S221A MEK1), as well as a constitutively active mutant of MEK1 to mimic (Fig. 5C; S217E/S221E MEK1), IL-1 stimulation of HK activity further supports the involvement of MEK in this response. The associated ability of a dominant interfering Ras mutant to abrogate this effect (Fig. 5B; K17N Ras) is also compatible with an upstream Ras-dependent coupling mechanism. To further evaluate the role of the classic MAPK pathway, we also directly tested the ability of IL-1 to activate ERK1/2. As shown in Fig. 6A, IL-1 increased ERK1/2 activity within 1 min, and this response was maximal at 15 min. The ability of IL-1 to activate ERK1/2 was completely blocked by pretreatment with 50 µM PD-98059 (Fig. 6B). In these experiments, MEK inhibition also abrogated basal ERK1/2 activity, and this effect was accompanied by small, statistically insignificant decreases in basal HK activity (Fig. 5, A and B; P > 0.33) that are consistent with our previous reports (14, 53) and suggest minor contributions of classic MAPK pathway activation to the maintenance of basal HK expression and activity in this model.
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induction of HK activity, we first examined the ability of pharmacological PKC inhibition by bisindolylmaleimide I to attenuate IL-1-stimulated HK activity. In a series of 24 experiments, 50 pM IL-1 increased total HK activity by nearly 40% (Fig. 7A; P < 0.0001). Pretreatment with 5 µM bisindolylmaleimide I for 1 h partially prevented this increase (Fig. 7A), but this effect did not achieve statistical significance and was mimicked by the inactive congener bisindolylmaleimide V under identical conditions. Neither bisindolylmaleimide had an independent effect on basal HK activity (Fig. 7A; P > 0.999). To further examine PKC involvement, we also tested cell-permeable PKC inhibitor peptides for the ability to attenuate IL-1-stimulated HK activity. In an independent series of nine experiments, 50 pM IL-1 increased HK activity significantly (Fig. 7B; P < 0.05 vs. unstimulated controls) in both the presence and absence of general PKC pseudosubstrate inhibitor peptides (30 µM myrPKC/20-28). These results are in good agreement with those obtained with the general pharmacological inhibitor bisindolylmaleimide I (Fig. 7A). Cell-permeable inhibitors of conventional PKC (myrPKCC2-4) and the novel PKC isoform (myrPKCV1-2) were similarly ineffective in attenuating IL-1-stimulated HK activity in this model (Fig. 7B). Finally, in another series of six experiments, IL-1 increased total HK activity by >50% (Fig. 7C), an effect that was not appreciably influenced by the antecedent expression of dominant interfering mutants of PKC (K368R), PKC (K436R), or PKC (K409R). Forced expression of a constitutively active PKC mutant (A159E) also failed to mimic the stimulatory effect of IL-1 (108 ± 8% vs. untransfected controls). Given the general failure of PKC antagonism to prevent IL-1 stimulation of HK activity, it is pertinent to note that initial attempts to demonstrate bisindolylmaleimide I attenuation of IL-1-stimulated ERK1/2 activation were also unsuccessful (data not shown), compatible with the contention that PKC does not play a major role in IL-1 activation of the classic MAPK pathway in this model.
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-stimulated HK activities were unaffected by the presence of the calcium chelator BAPTA at concentrations as high as 10 µM (data not shown). These findings are compatible with the inability of calcium ionophores to increase HK activity in this cell type in the presence of normal extracellular calcium (14). They are also compatible with our inability to demonstrate IL-1-mediated changes in intracellular Ca2+ concentration in the present model (Robey RB and Hecquet C, unpublished observation).
Although not a classic feature of IL-1 signaling, PTX has been reported to disrupt IL-1 signaling in some cells (45), and partial inhibition of IL-1 action by PTX has been reported in cultured mesangial cells (56). The type I IL-1 receptor possesses only a single transmembrane domain and, hence, does not conform to the classic heptaspanning G protein-coupled receptor paradigm. Nevertheless, the reported sensitivity to PTX has been interpreted as evidence of coupling between IL-1 signaling and PTX-sensitive G protein activation (45). We have previously reported G protein-coupled stimulation of HK activity in mesangial cells (14, 55), but PTX sensitivity has not been demonstrated for any of these effects. Similarly, 0.1 µg/ml PTX failed to prevent stimulation of total HK activity by 50 pM IL-1 at 24 h (154 ± 17 vs. 166 ± 13% of control values in the presence and absence of PTX, respectively), so this issue was not examined further.
IL-1-stimulated HK activity is associated with a selective increase in HKII isoform abundance.
We previously demonstrated (14, 54) the presence of all three high-affinity renal HK isoforms (HKI, HKII, and HKIII) in cultured mesangial cells. We also reported general requirements for both ongoing gene expression and de novo protein synthesis in the stimulation of HK activity by phorbol esters and thrombin and speculated that increased HK expression may contribute to these effects (53, 55). The presence of all three HK isoforms was confirmed in immunoblots of cell lysates from both IL-1-stimulated cells and unstimulated control cells (Fig. 8A). As depicted in Fig. 8B, IL-1 (50 pM) increased HKII isoform abundance more than twofold within 24 h, whereas the abundances of the HKI and HKIII isoforms were not similarly affected. MEK inhibition by pretreatment with 50 µM PD-98059 prevented the increase in HKII abundance (Fig. 8C), suggesting a causal relationship between ERK1/2 activation (Fig. 6) and the subsequent increase in HK activity.
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increased both net Glc utilization (3.4 ± 0.3 vs. 2.8 ± 0.3 mmol·g–1·6 h–1; P < 0.01) and net lactate accumulation (5.4 ± 0.3 vs. 4.4 ± 0.2 mmol·g–1·6 h–1; P < 0.01) within 24 h of stimulation. As reported previously (53–55) for both phorbol ester- and thrombin-stimulated Glc metabolism in this cell type, the stoichiometric ratio of lactate accumulation to Glc disappearance in the medium was consistently between 1.6 and 1.7 for both stimulated and unstimulated cells (data not shown), which does not suggest that metabolic uncoupling contributes to these changes. |
DISCUSSION |
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MEK ERK), as well as an associated increase in HKII isoform abundance, suggests specific underlying mechanisms for this response in mesangial cells (Fig. 9).
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IL-1 and IL-1 are structurally related proinflammatory cytokines produced by a variety of cell types (47). These factors exert a broad range of biological actions (16), including growth factor-like properties such as the stimulation of cellular proliferation (61). Of particular relevance to the present work, IL-1 production has been described for intrinsic glomerular endothelial (63), epithelial (61), and mesangial (36, 38, 63, 65) cells. This suggests the possibility of both autocrine and paracrine regulation of HK activity by IL-1 within the glomerulus. Activated monocytes and macrophages represent another important source of IL-1 cytokines (37, 38), and an association of both immune (63) and nonimmune (15, 40, 64) experimental models of glomerular disease, including diabetes (69), with glomerular macrophage infiltration suggests that these responses have pathophysiological relevance.
The IL-1 receptor antagonist, IL-1ra, constitutes the third known member of the IL-1 gene family (71). This factor exhibits significant structural homology with both IL-1 and IL-1 and has a similar affinity for the type I IL-1 receptor expressed by mesangial cells (71). However, in contrast to the other IL-1 family members, IL-1ra is incapable of receptor activation. IL-1ra thus directly competes with both IL-1 and IL-1 for receptor binding and functions as a naturally occurring peptide antagonist of IL-1 action (16, 18, 21). The IC50 of the recombinant protein employed in the present studies is typically in the 30–60 ng/ml range according to the supplier's specifications (R&D Systems). Thus the apparent IC50 of 89 ng/ml reported in this study is compatible with a direct effect at the level of the type I receptor.
Both secretory phospholipase A2 (sPLA2) and its reaction product, LPA, are capable of increasing mesangial cell HK activity and HKII isoform expression in mesangial cells (14). It is therefore of considerable interest that IL-1 has been shown to stimulate sPLA2 release by a variety of cell types, including mesangial cells (51, 52, 58). Although this raises the intriguing possibility that these diverse stimuli share a common proximal mechanism of classic MAPK pathway activation, a demonstrated requirement for PKC activation by LPA (14), but not by IL-1, argues against this possibility. It is more likely that IL-1, like epidermal growth factors (54), activates the classic MAPK module (Raf MEK ERK) distal to PKC, presumably via a Ras-dependent mechanism (Fig. 9).
The suggestion that HKII constitutes the principal inducible HK isoform in this cell type is also of considerable interest, given the fact that HKII represents the major inducible isoform in the insulin-sensitive peripheral tissues affected by diabetes (48). However, several features distinguish HK regulation in mesangial cells from that observed in other end-organ targets of this disease. First, the lack of responsiveness to insulin or insulin-like growth factors (Ref. 54; Robey, unpublished observations) is consistent with the known insulin resistance of renal Glc metabolism but constitutes a fundamental difference in regulation. Second, there appears to be a uniform requirement for classic MAPK pathway activation (14, 53–55). Finally, calcium appears to be dispensable for HKII induction in mesangial cells. These features contrast markedly with the corresponding regulatory behavior described in skeletal myotubes (28, 49). When combined with previous observations that mesangial cell HK activity is not appreciably affected by factors known to increase HK activity in muscle or adipose (54, 55), these findings suggest cell type-specific differences in HKII regulation and at least two distinct pathways of HKII induction.
We conclude that IL-1 cytokines constitute novel regulators of HK activity in mesangial cells. This regulation requires signal transduction via type I IL-1 receptors and the classic MAPK pathway, a process that appears to be Ras dependent. Associated selective increases in HKII isoform abundance suggest a causal relationship with increased total HK activity. The observed sensitivity of these responses are also consonant with the reported EC50 of the IL-1 cytokines used in these studies (1–20 pg/ml; Sigma), suggesting physiological relevance. Because reports of altered HK activity in the adult kidney have been largely restricted to pathological conditions associated with renal functional or structural abnormalities, our findings have pathophysiological as well as physiological implications. They also suggest specific mechanisms whereby mesangial cell Glc metabolism may be coupled to glomerular injury. We have previously suggested (14, 53–55) that such changes may constitute an important general adaptive response to cellular injury. Although not directly addressed in the present study, indirect support for such a role may be found in the reported antiapoptotic effects of HKs (4, 10, 24, 25, 42, 50). The ability of IL-1 to attenuate pulmonary injury in an in vivo oxidative stress model (66) associated with adaptive increases in HKII expression (2) is clearly compatible with this contention, as is the corresponding ability of forced HKII expression to protect cultured lung epithelial cells against oxidant injury (1). Additional support may be found in recent genetic evidence linking a noncoding IL-1 gene polymorphism with increased cytokine expression that inversely correlated with risk for the development of end-stage renal disease (7). Further studies are needed to establish the precise role of such changes in mesangial cells, but our findings clearly validate the importance of classic MAPK pathway activation in mesangial cell HK regulation and suggest a novel mechanism for coupling metabolism to inflammation and cellular injury.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* N. Taneja and P. E. Coy contributed equally to this work. 
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