Hypoxia-specific upregulation of calpain activity and gene expression in pulmonary artery endothelial cells

Jianliang Zhang, Jawaharlal M. Patel, and Edward R. Block

Department of Medicine, University of Florida, and Medical Research Service, Veterans Affairs Medical Center, Gainesville, Florida 32608-1197

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The effects of exposure to hypoxia on the catalytic activity and mRNA expression of calpain, a calcium-regulated neutral cysteine protease, were examined in porcine pulmonary artery endothelial cells (PAECs). Specificity of the response to hypoxia was determined by comparing the effects of hypoxic exposure with exposure to oxidants such as nitrogen dioxide (NO2) and nitric oxide (NO), as well as to the sulfhydryl reactive chemical acrolein. Exposure of cells to hypoxia (0% O2) for 1 and 12 h significantly increased catalytic activity (P < 0.01 for both 1 and 12 h vs. control cells), as well as mRNA expression (P < 0.01 for 1 h and P < 0.05 for 12 h vs. control cells) of calpain. With more prolonged exposure to 24 h of hypoxia, calpain activity remained significantly elevated, whereas calpain mRNA expression returned to the control level. Calpain activities in cells exposed to NO2 [5 parts/million (ppm)] or NO (7.5 ppm) for 1 h or to acrolein (5 µM) for 1 and 24 h were unchanged. However, calpain activities in cells exposed to NO2 or NO for 24 h were significantly (P < 0.05) reduced compared with control cells. The hypoxia-induced increases in calpain mRNA content were prevented by the transcriptional inhibitor actinomycin D and by calpain inhibitor I. In addition, hypoxia increased the degradation of nuclear factor-kappa B (NF-kappa B) inhibitor Ikappa B and enhanced the translocation of the p50 subunit of NF-kappa B to the nuclear membrane. Pretreatment with the calpain-specific inhibitor E-64d prevented hypoxia-induced mRNA expression and degradation of Ikappa Balpha , as well as translocation of p50 subunit to the nuclear membrane. These results demonstrate for the first time that hypoxia upregulates calpain activity and mRNA expression in PAECs and that the upregulation is specific to hypoxia. Upregulation appears to involve activation of the transcription factor NF-kappa B.

endothelium

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
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References

ENDOTHELIAL CELLS form a continuous monolayer on the luminal surface of the vasculature and provide a metabolically active, thromboresistant, and semipermeable barrier that regulates transport of water, solutes, and particulate material between blood and tissues (30). Numerous studies demonstrate that exposure to hypoxia results in significant alterations in the structure and function of endothelial cells in vivo (37, 39) and in vitro (6, 18, 26, 46). Bhat and Block (5), Block et al. (6), and others (8, 11) have described dramatic reductions in membrane phospholipids in a variety of mammalian cells, including lung endothelial cells exposed to hypoxic conditions. The hypoxia-induced alterations in phospholipid metabolism in lung endothelial cells are due in large part to increased cytosolic free calcium, which results in activation of the phospholipid deacylation pathway that involves calcium-dependent enzymes such as phospholipase A2 (5, 8, 11, 35).

Several recent reports suggest that another calcium-dependent enzyme, calpain, plays a critical role in hypoxia-induced cell injury in rat renal proximal tubular cells (10) and rat cardiac myocytes (14). Calpain, which was initially described in 1964 (13), is the name given to a family of cytosolic, calcium-activated, neutral proteases that, for the most part, are ubiquitous (31, 42). Substrates for this family of enzymes include the cytoskeletal proteins fodrin, talin, and filamin, microtubule-associated proteins, and a number of membrane proteins, including growth factor receptors [e.g., epidermal growth factor (EGF)], adhesion molecules (e.g., integrin, cadherin), and ion transporters (e.g., Ca2 +-ATPase) (31, 42). Su and Block (41) recently reported that calpain activity is increased in lung endothelial cells exposed to hypoxia and that the increased calpain activity plays a critical role in the mobilization of L-arginine from proteins in these hypoxic cells.

There are two major isoforms of calpain: calpain I (also known as µ-calpain), which binds calcium with relatively high affinity, and calpain II (also known as m-calpain), which binds calcium with relatively low affinity. Both isoforms are heterodimers, consisting of a larger (~80 kDa) catalytic subunit and a smaller (~30 kDa) subunit that helps regulate its activity. Calpain exists in the cytosol as the inactive proenzyme procalpain, which translocates from the cytosol to the cell membrane in the presence of micromolar levels of calcium. Autocatalytic activation of procalpain to active calpain occurs at the membrane in the presence of physiological levels of calcium and phosphatidylinositol (31, 42). Autolysis of the catalytic subunit is an essential criterion for in vivo proteolytic activity (9, 31).

A number of investigators (16, 17, 21-24, 38) have reported that hypoxia upregulates the expression of a variety of genes in endothelial cells. The mechanism responsible for this hypoxic upregulation is not known. However, it is known that oxidative stress and reactive oxygen intermediates increase the transcription of a variety of genes by activating the transcription factor nuclear factor-kappa B (NF-kappa B) (34, 36, 40). A characteristic feature of NF-kappa B is its activation by posttranslational mechanisms that involve dissociation of its inhibitory protein Ikappa B. The release of other subunits of NF-kappa B, namely p50 and p65, from Ikappa B permits the translocation of p50 and p65 into the nucleus, where they bind to DNA enhancer motifs and regulate the transcription of a diverse series of genes (25, 43). In the present study, we examined the mechanism by which hypoxia upregulates calpain activity in lung endothelial cells and, in particular, whether hypoxia regulates calpain gene expression through the activation of transcription factor NF-kappa B.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals. A PolyATract mRNA isolation system was purchased from Promega (Madison, WI). Calpain inhibitor I was acquired from Calbiochem (La Jolla, CA). Resorufin-labeled casein, digoxigenin-labeled deoxyuridine triphosphates (DIG-dUTPs), calpain-specific inhibitor E-64d, and Genius labeling and detection kits were obtained from Boehringer Mannheim (Indianapolis, IN). Dithiothreitol, Nonidet P-40, antipain, aprotinin, bestatin, and phosphoramidon were obtained from Sigma (St. Louis, MO). NO2 and NO premixed gases were obtained from Air Products (Jacksonville, FL). All other chemicals were obtained from Fisher Scientific (Orlando, FL).

Cultured cells. Endothelial cells were obtained from the main pulmonary artery of 6- to 7-mo-old pigs and were propagated in monolayer cultures and characterized as described by Patel and Block (27). Fifth- to seventh-passage cells in postconfluent monolayers maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 4% fetal bovine serum (HyClone Labs, Logan, UT) and antibiotics (100 U/ml of penicillin, 100 µg/ml of streptomycin, 20 µg/ml of gentamicin, and 2 µg/ml of Fungizone) (maintenance medium) were used in all studies.

Exposure of cells. Confluent monolayers of pulmonary artery endothelial cells (PAECs) in RPMI 1640 were exposed to 1) 0% O2-5% CO2-95% N2 (hypoxia), 2) 5 parts/million (ppm) NO2 in air containing 5% CO2, 3) 7.5 ppm NO in 5% CO2-95% N2, or 4) 5 µM acrolein (ACR) in Hanks' balanced salt solution (HBSS) at 37°C for 1-24 h, as previously reported (6, 20, 27, 28). Control cells were exposed to air containing 5% CO2 or HBSS alone (ACR controls) under identical conditions to the respective exposure protocol. In some experiments, cells were pretreated with actinomycin D (0.2 µM), calpain inhibitor I (100 µM), or E-64d (20 µg/ml) for 30 min at 37°C followed by exposure to hypoxia for 1 h at 37°C. After exposure, cells were used for measurement of calpain activity, calpain-specific mRNA expression, and isolation of cytosolic and nuclear fractions.

Measurement of calpain activity. Calpain activity was measured by monitoring the absorbance change of resorufin-labeled casein as a substrate according to Twining (44). After exposure, PAEC monolayers were rinsed, scraped, and homogenized in extraction buffer (10 mM Tris · HCl, pH 7.5). The homogenate was then centrifuged at 14,000 rpm for 10 min, and 100 µl of the supernatant were mixed with 50 µl of substrate solution (0.4% resorufin-labeled (wt/vol) casein in double-distilled water) and 50 µl of incubation buffer I (200 mM Tris · HCl, pH 8.0, 20 mM CaCl2, and 10 mM cysteine) or incubation buffer II (200 mM Tris · HCl, pH 8.0, 4 mM EDTA, and 80 mM EGTA). The reaction mixture was kept at 37°C for 30 min, and the reaction was stopped by adding 480 µl of 5% trichloroacetic acid. After centrifugation at 14,000 rpm for 5 min, 400 µl of supernatant were mixed with 600 µl of assay buffer (0.5 M Tris · HCl, pH 9.0), and the absorbance at 574 nm was read at room temperature. Values from samples incubated with buffer II (containing EDTA + EGTA but no cysteine or CaCl2) were subtracted from those incubated with buffer I (containing cysteine and CaCl2). Thus this in vitro method measures calcium-dependent calpain activity, which represents 80% of the total activity. Although the substrate used in this assay can also be used by other proteolytic enzymes such as cathepsins (32), our experimental conditions ensure specificity for calpain. For example, cathepsins are inactive at pH > 7.0, and their catalytic activity is exclusively calcium independent (4). One unit of calpain activity was defined as the amount of enzyme per 1 × 106 cells that increased the absorbance 0.1 units at 574 nm during the 30-min incubation period.

Rapid amplification of the 3' cDNA end of porcine calpain. The 3'-end of porcine calpain cDNA was isolated from a porcine PAEC cDNA library (48) by performing PCR with an adapter primer (AP1) and a sense gene-specific primer (5'-ACC TTC GAG CCC AAC AAG GAC GGG GAC-3') (Expand Long Template PCR System, Boehringer Mannheim). The gene-specific primers were designed based on a conserved region of the human calpain II cDNA sequence (15) for calcium binding. PCR was carried out for 30 cycles under the following conditions: denature at 94°C for 30 s and anneal and extend at 68°C for 6 min on a thermal cycler (Perkin-Elmer DNA thermal cycler 480, Foster City, CA). After gel purification, the 3'-rapid amplification of cDNA end (RACE) fragment was cloned into pGEM-T vector (Promega) and was sequenced by the Interdisciplinary Center for Biotechnology Research at the University of Florida (Gainesville, FL). The sequence of the isolated porcine calpain 3'-end fragment is 87% identical to human calpain II and 62% identical to human calpain I (1).

mRNA isolation and Northern analysis. Total poly(A)+ RNA was extracted directly from 5 × 107 cells with the PolyATract mRNA isolation system following the manufacturer's instructions. The glyoxal-denatured mRNA (~5 µg/sample) and digoxigenin-labeled RNA molecular-weight marker II (1.6-7.4 kb; Boehringer Mannheim) were fractionated on a 0.8% (wt/vol) agarose gel before being blotted onto a nylon membrane (Zeta-Probe GT, Bio-Rad, Richmond, CA). Membranes were prehybridized in DIG-Easy-Hyb solution at 43°C for 1 h. The porcine calpain II cDNA probe was labeled with digoxigenin-dUTP by the random-primer method (12). The labeled probe (1 µg) was denatured by boiling for 5 min and was added to 50 ml of DIG-Easy-Hyb solution. Hybridization was carried out at 43°C for 16 h. After hybridization, the blot was washed twice in 2× SSC (saline-sodium citrate; 10× SSC = 1.5 M NaCl and 0.15 M sodium citrate)-0.1% SDS for 5 min at room temperature and with 1× SSC-0.1% SDS at 65°C for 25 min. The hybridized probes were immunodetected with anti-digoxigenin-AP and the chemiluminescence substrate CSPD (Genius Labeling and Detection Kits, Boehringer Mannheim), as described by the manufacturer, and were then exposed to Kodak XAR-5 film for 30-60 min. To document the amount of RNA loaded, the same blot was stripped twice in 0.1× SSC-0.1% SDS at 65°C for 1 h. The 18S complementary oligonucleotide (-ACG GTA TCT GAT CGT CTT CGA ACC-) (47) was labeled at its 3'-end with terminal transferase by synthesis of a DNA tail of 40-50 nucleotide lengths with several incorporated DIG-dUTPs, as described previously (33). Membranes were hybridized at 50°C overnight and washed twice at 65°C in 1× SSC-0.1% SDS for 25 min. The immunochemiluminescent method was used to detect the hybrids under the conditions described above. Quantification of the porcine calpain mRNA was performed with a laser densitometer (Ultroscan XL, LKB, Bromma, Sweden). The level of the calpain mRNA was standardized to the 18S mRNA contents.

Isolation of nuclear and cytosolic fractions. Nuclear and cytosolic fractions were prepared from the cells exposed to hypoxia or normoxia, as previously described (3). Briefly, cells from 6 × 100-mm dishes were harvested, washed twice in cold PBS, and resuspended in 200 µl of lysing buffer (10 mM HEPES buffer, pH 7.9, 60 mM KCl, 1 mM dithiothreitol, 0.2% vol/vol Nonidet P-40, 74 µM antipain dihydrochloride, 0.1 µM aprotinin, 130 µM bestatin, 50 µM chymostatin, 1 µM E-64d, 1 mM EDTA, 1 µM leupeptin, 1 mM Pefabloc SC, 1 µM pepstatin, and 200 nM phosphoramidon) for 5 min. Samples were centrifuged at 1,000 g for 5 min, and then the supernatant (cytosolic fraction) was stored at -80°C until use. Pelleted nuclei were gently rinsed twice with 1 ml of Nonidet P-40-free lysing buffer and resuspended in 300 µl of the same buffer. The resuspended nuclei were layered on top of 300 µl of Nonidet P-40-free lysing buffer containing 30% saccharose. After centrifugation at 5,000 g for 10 min, the supernatant was gently removed, and the pelleted nuclei were resuspended in 200 µl of nuclear suspension buffer (250 mM Tris · HCl, pH 7.8, 60 mM KCl, 1 mM dithiothreitol, 74 µM antipain dihydrochloride, 0.1 µM aprotinin, 130 µM bestatin, 50 µM chymostatin, 1 µM E-64d, 1 µM leupeptin, 1 mM Pefabloc SC, 1 µM pepstatin, and 200 nM phosphoramidon). A small aliquot of the nuclear preparation was stained with trypan blue and observed by light microscopy to verify the integrity of the nuclei. The nuclear suspension was then subjected to three cycles of freezing and thawing. The suspension was cleared by centrifugation at 14,000 g for 15 min, and the supernatant was collected and stored at -80°C until use.

Western analysis of p50 and Ikappa Balpha proteins. The proteins (15-20 µg) from cytosol and nuclei were fractionated on a 7.5% SDS-PAGE gel and were blotted onto nitrocellulose membranes (Bio-Rad). The blots were incubated in blocking solution (0.2% dry milk in TBS-T; TBS-T = 0.1% Tween 20, 10 mM Tris · HCl, pH 7.5, and 100 mM NaCl) and then hybridized with anti-p50 or anti-Ikappa B-alpha polyclonal antibody (Santa Cruz Biotechnology; 1:1,000 diluted in TBS-T) at room temperature for 3.5 h. After being washed, the membranes were incubated in 1:2,000 diluted anti-IgG, alkaline phosphatase- linked whole antibody (Bio-Rad) for 45 min. The immunoreactive bands were visualized by enhanced chemiluminescence reagents with Kodak X-OMAT films. The blots were scanned with a laser densitometer (Ultroscan XL, LKB) to quantify p50 or Ikappa Balpha protein contents.

Statistical analysis. Within each experiment, control and treated cells were matched for cell line, age, number of passages, and number of days postconfluence. Statistical significance for the effects of treatment (e.g., hypoxia) on the catalytic activity and steady-state mRNA levels of calpain as well as on p50 and Ikappa B contents was determined by analysis of variance and Student's t-test (45).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The effect of hypoxia on calpain activity in PAECs. Exposure to hypoxia for 1-24 h significantly (P < 0.01 vs. control cells) increased calpain activity in porcine PAECs (Fig. 1). The maximal increase in calpain activity was observed in 1-h hypoxia-treated cells and was almost twice that in the control cells. Calpain activity remained significantly elevated after 12- and 24-h exposures to hypoxia.


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Fig. 1.   Effect of exposure to hypoxia on calpain activity in porcine pulmonary artery endothelial cells (PAECs). Intact cell monolayers were exposed to normoxia or hypoxia for 1-24 h. After exposure, calpain activities in cell homogenates were measured as described in MATERIALS AND METHODS. Data are means ± SE; n = 10 experiments. * P < 0.01 vs. normoxic control cells exposed for same duration.

Effect of NO, NO2, and ACR on calpain activity in PAECs. To determine whether the effects of hypoxia on calpain activity were specific, calpain activity was measured in PAECs exposed to NO, NO2, or ACR for 1 or 24 h. No significant changes in calpain activity were observed in cells exposed to NO, NO2, or ACR for 1 h (data not shown). However, calpain activity in cells exposed to NO or NO2 for 24 h was decreased ~25% (P < 0.05 each) compared with control cells. Exposure to 5 µM ACR for 24 h had no effect on calpain activity (Fig. 2).


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Fig. 2.   Effects of NO, NO2, and acrolein (ACR) on calpain activity in porcine PAECs. After exposure of cell monolayers to NO, NO2, or ACR (solid bars) or their normoxic control cells (open bars) for 24 h, calpain activity was measured as in MATERIALS AND METHODS. Data are means ± SE; n = 6 experiments. * P < 0.01 vs. control cells.

Effect of hypoxia on calpain mRNA levels in PAECs. To evaluate whether hypoxia-induced modulation of calpain activity is regulated at the transcriptional level, calpain mRNA contents were measured in control and hypoxia-treated cells. The porcine calpain cDNA probe was used to detect the mRNAs for calpain in porcine PAECs exposed to normoxia and hypoxia for 1-24 h. Exposure of PAECs to hypoxia increased steady-state mRNA levels of calpain (Fig. 3A). When data were normalized to 18S mRNA, calpain mRNA contents were found to be increased twofold (P < 0.01) in cells exposed to hypoxia for 1 h and nearly 40% in cells exposed for 12 h (Fig. 3B). Calpain mRNA content in cells exposed to hypoxia for 24 h was comparable to that in control cells.


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Fig. 3.   Effect of hypoxia on calpain mRNA levels in PAECs. After cell monolayers were exposed to hypoxia for 1-24 h, poly(A)+ RNAs were extracted from these cells. Denatured mRNAs (~5 µg/lane) were fractionated and blotted onto a nylon membrane. Membrane was hybridized with porcine calpain cDNA probe labeled with digoxigenin-dUTP by random-primer method as described in MATERIALS AND METHODS. To quantify changes in mRNA levels, the same blot was stripped and probed with an 18S complementary oligonucleotide and then density of Northern blots was measured. A: representative Northern blot of mRNA isolated from porcine PAECs. Lane 1, control cells; lane 2, 1 h of hypoxia; lane 3, 12 h of hypoxia; lane 4, 24 h of hypoxia. B: levels of calpain mRNA (means ± SE) standardized to 185 S mRNA contents from 3 experiments of corresponding blots shown in A. * P < 0.01 vs. control cells. ** P < 0.05 vs. control cells.

Effect of NO, NO2, and ACR on calpain mRNA content in PAECs. To determine whether the early hypoxia-induced upregulation of calpain mRNA levels is specific to decreased O2 tension, porcine PAECs were exposed to NO, NO2, or ACR, after which steady-state calpain mRNA contents were quantitated by Northern blot analysis. As shown in a representative autoradiograph (Fig. 4A) and in densitometric analysis of data from three experiments (Fig. 4B), the calpain mRNA contents in the cells exposed to NO, NO2, and ACR were comparable to control cells.


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Fig. 4.   Effects of NO, NO2, and ACR on calpain mRNA in PAECs. After cell monolayers were exposed to NO, NO2, or ACR for 1 h, poly(A)+ RNAs were extracted from these cells. Denatured mRNAs (~5 µg/lane) were fractionated and blotted onto a nylon membrane. Membrane was hybridized with porcine calpain cDNA probe as described in MATERIALS AND METHODS. To quantify changes in mRNA levels, the same blot was stripped and reprobed with an 18S complementary oligonucleotide, and then density of Northern blots was scanned. A: representative Northern blot of mRNA isolated from porcine PAECs. Lane 1, control cells; lane 2, 1-h exposure to NO; lane 3, control cells; lane 4, 1-h exposure to NO2; lane 5, control cells; lane 6, 1-h ACR treatment. B: levels of the calpain mRNA (means ± SE) standardized to 18S mRNA contents from 3 experiments of corresponding blots shown in A.

Effect of actinomycin D on hypoxia-induced increases in calpain mRNA contents. To elucidate whether hypoxia-induced upregulation of calpain gene expression in PAECs is related to transcription, cells were preincubated with actinomycin D for 30 min and then exposed to hypoxia for 1 h. The calpain mRNA contents were comparable in actinomycin D-treated cells incubated under normoxic or hypoxic conditions, whereas exposure to hypoxia in the absence of actinomycin D increased calpain mRNA contents nearly twofold compared with cells exposed to normoxia alone (Fig. 5). These results indicate that transcription is required for the hypoxic induction of calpain mRNA in PAECs.


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Fig. 5.   Effect of actinomycin D on hypoxia-induced increase in calpain mRNA contents in PAECs. After exposure, Northern blot analysis of calpain mRNA was carried out as described in MATERIALS AND METHODS. A: representative blot of cultured cells exposed to normoxia (lane 1) or hypoxia (lane 2) for 1 h. Some cell monolayers were incubated for 30 min with actinomycin D (0.2 µM) and then exposed to normoxia (lane 3) or hypoxia (lane 4) for 1 h. B: levels of the calpain mRNA (means ± SE) standardized to 18S mRNA contents. * P < 0.01 vs. normoxia control cells; n = 3 experiments of corresponding blots shown in A.

Effect of actinomycin D on hypoxia-induced increases in calpain activity. To verify that actinomycin D also prevents the hypoxia-induced increase in calpain activity in PAECs, cells were preincubated with actinomycin D for 30 min and then exposed to hypoxia for 1 h, after which calpain activities were determined. As expected, calpain activity was increased in cells exposed to hypoxia in the absence of actinomycin D (Fig. 6). However, the hypoxia-induced increase in calpain activity, like the hypoxia-induced increase in calpain mRNA, was prevented by incubation with actinomycin D. 


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Fig. 6.   Effect of actinomycin D on hypoxia-induced increases in calpain activity in PAECs. Intact cell monolayers were exposed to normoxia (lane 1) or hypoxia (lane 2) for 1 h. Some cell monolayers were incubated for 30 min with actinomycin D (0.2 µM) and then exposed to normoxia (lane 3) or hypoxia (lane 4) for 1 h. After exposure, calpain activities in cell homogenates were measured as described in MATERIALS AND METHODS. Data are means ± SE (n = 6). * P < 0.01 or ** P < 0.05 vs. normoxic control cells exposed for same duration.

Effects of calpain inhibitors on the hypoxia-induced increase in calpain mRNA contents in PAECs. To examine whether calpain itself is involved in the hypoxia-induced upregulation of calpain mRNA contents, studies were conducted in cells exposed to normoxia or hypoxia in the absence or presence of calpain inhibitor I and calpain-specific inhibitor E-64d in the medium. As shown in Fig. 7, in the absence of calpain inhibitor I, calpain mRNA levels were increased in hypoxia-treated cells. However, in the presence of calpain inhibitor I, the mRNA contents were comparable in normoxic and hypoxia-treated cells. Calpain inhibitor I alone had no effect on calpain mRNA (Fig. 7). Similar effects on mRNA contents were observed in the presence and absence of E-64d (data not shown). These results are comparable to the effects of actinomycin D. 


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Fig. 7.   Effect of a calpain inhibitor on the hypoxia-induced increase in calpain mRNA contents in PAECs. After exposure, Northern blot analysis of calpain mRNA was carried out as described in MATERIALS AND METHODS. A: representative blot of cultured cells exposed to normoxia (lane 1) or hypoxia (lane 2) for 1 h. Some cell monolayers were incubated for 30 min with calpain inhibitor I (100 µM) and then exposed to normoxia (lane 3) or hypoxia (lane 4) for 1 h. B: levels of the calpain mRNA (means ± SE) standardized to 18S mRNA contents. * P < 0.01 vs. normoxia control cells (lane 1); n = 3 experiments of corresponding blots shown in A.

Effects of hypoxia on transcription factor NF-kappa B. Because calpain inhibitor I blocks activation of NF-kappa B (21) and because calpain inhibitor I E-64d and actinomycin D prevent hypoxia-induced increases in calpain mRNA expression, we examined whether hypoxia modulates activation of the transcription factor NF-kappa B. As shown in Fig. 8, the NF-kappa B inhibitor Ikappa Balpha is decreased in the cytosol of cells exposed to hypoxia (P < 0.01). However, Ikappa Balpha levels in cytosolic fractions of E-64d-treated control and E-64d-treated hypoxia-exposed cells were comparable and were significantly increased compared with the untreated control cells (P < 0.01). Nuclear fraction Ikappa Balpha levels were comparable in control (untreated), hypoxia, E-64d, and E-64d and hypoxia groups and were <5% of cytosolic fraction Ikappa Balpha levels in all groups (not shown). In contrast, exposure to hypoxia significantly (P < 0.01) increased translocation of the NF-kappa B subunit p50 from the cytosolic fraction to the nuclear fraction in PAECs (Fig. 9). Although E-64d did not alter the p50 content of the nuclear membrane, the hypoxia-induced increase in p50 content of the nuclear membrane was prevented by E-64d in PAECs (Fig. 9). These results indicate that activation of the transcription factor NF-kappa B is responsible for hypoxia-induced calpain gene expression in porcine PAECs.


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Fig. 8.   Degradation of nuclear factor-kappa B (NF-kappa B) inhibitor Ikappa Balpha in porcine PAECs exposed to normoxia and hypoxia. Cytosolic fraction was isolated from PAECs pretreated with and without calpain-specific inhibitor E-64d and exposed to normoxia or hypoxia for 1 h. Fractions (40 µg) were separated by 12% SDS-PAGE gels and immunoblotted with Ikappa Balpha antibody as in MATERIALS AND METHODS. A: representative immunoblot. Lane 1, control cells; lane 2, exposed to hypoxia; lane 3, E-64d control cells; lane 4, E-64d + hypoxia. B: Ikappa B contents (means ± SE of original densitometry scoring data) from 3 experiments of corresponding blots shown in A. * P < 0.01 vs. cystolic control.


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Fig. 9.   Subcellular location of the NF-kappa B subunit p50 in porcine PAECs exposed to normoxia and hypoxia. Nuclear extract was isolated from PAECs pretreated with or without E-64d and exposed to normoxia or hypoxia for 1 h. Extracts (40 µg) were separated by 12% SDS-PAGE gels and immunoblotted with p50 antibody as described in MATERIALS AND METHODS. A: representative immunoblot. Lane 1, control cells; lane 2, exposed to hypoxia; lane 3, E-64d control; lane 4, E-64d + hypoxia. B: p50 subunit contents (means ± SE of original densitometry scoring data) from 3 experiments of corresponding blots shown in A. * P < 0.01 vs. cytosolic control.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously reported that the activity of calpain, a calcium-dependent neutral cysteine protease, is increased in porcine PAECs exposed to hypoxia (41). The mechanism responsible for this increase in calpain activity in hypoxic PAECs has not been defined. Because exposure to hypoxia has been reported to increase intracellular calcium in endothelial cells (2), it has been assumed that this is the mechanism by which hypoxia activates calpain. However, the present study demonstrates that exposure to hypoxia for as little as 1 h increases both calpain activity and mRNA, and the magnitude of the increase in activity and in mRNA is comparable. Moreover, the increases in calpain mRNA and activity can be blocked by actinomycin D, an inhibitor of transcription, indicating that acute hypoxia upregulates calpain activity through a transcriptional process in porcine PAECs. Other agents known to injure lung endothelial cells and to increase intracellular calcium content, such as NO, NO2, and ACR, had no effect on (or decreased) calpain activity and had no effect on calpain mRNA content. These observations suggest that calpain mRNA upregulation by acute hypoxia is specific and may play a role in the initial stages of hypoxic injury in porcine PAECs or in the early response to such injury. Edelstein et al. (10) found that calpain activity increased in hypoxic rat proximal tubule cells before evidence of plasma membrane damage, leading these authors to hypothesize that calpain plays a role in hypoxic injury of the rat proximal tubule rather than being the result of the injury. More prolonged exposure of our porcine PAECs to hypoxia, i.e., 24 h, was accompanied by a continued increase in calpain activity but the return of calpain mRNA content to control levels. The reason for this discordance between activity and message in cells exposed to hypoxia for 24 h is not known, but several possibilities exist. First, the half-life of the calpain molecule may be much longer than the half-life for the transcript. As such, the increase in calpain activity would be expected to outlast the increase in calpain mRNA content. Second, the persistent increase in calpain activity in cells exposed to hypoxia for 24 h may be due to elevated intracellular calcium and phosphatidylinositol contents in these cells, leading to continued activation of procalpain, a mechanism that is independent of the transcriptional upregulation of calpain occurring with acute exposure to hypoxia (31, 42).

The hypoxia-induced increase in calpain expression observed in the present study appears to be associated with activation of the transcription factor NF-kappa B. NF-kappa B-activated gene expression requires dissociation and/or proteolysis of the inhibitor protein Ikappa Balpha in the cytosol followed by translocation of the NF-kappa B dimer (p50-p65) to the nucleus. The results of the present study are consistent with this mechanism in that hypoxia increased the degradation of Ikappa Balpha in the cytosol and increased the translocation of the p50 subunit to the nucleus in hypoxia-exposed cells. The precise mechanism by which hypoxia increased the dissociation and/or degradation of Ikappa Balpha in the cytosol in the present study is not known. However, hypoxia is known to activate a signaling mechanism that increases phosphorylation of proteins (7) and also activates a number of proteolytic enzymes including calpain (10, 41). Our results, which demonstrate that calpain inhibitor I and E-64d prevent the hypoxia-induced upregulation of calpain mRNA degradation of Ikappa Balpha and activation of NF-kappa B, indicate that hypoxia activates calpain, which, in turn, cleaves Ikappa B from NF-kappa B. This is consistent with one recent report (22) suggesting that calpain inhibitors I and II block the degradation of Ikappa Balpha and the activation of NF-kappa B. Although calpain inhibitor I is nonspecific, similar results obtained with E-64d confirm that hypoxia-induced calpain gene expression is regulated by NF-kappa B.

In conclusion, our results reveal that increased mRNA expression and catalytic activity of calpain are specific to hypoxia in lung endothelial cells. The hypoxia-induced increased expression of calpain is mediated via transcriptional mechanisms that may involve activation of the transcription factor NF-kappa B. Activation of NF-kappa B has significant implications for cell physiology because NF-kappa B regulates the expression of a variety of genes and therefore proteins in mammalian cells (43) and may be a mechanism by which hypoxia regulates gene expression in endothelial cells. Because several integral membrane proteins, as well as cytoskeletal proteins, are calpain substrates, hypoxia-induced expression of calpain may also play a role in the loss of membrane integrity and cellular architecture that are early features of hypoxic injury in endothelial cells (4, 6, 19, 29).

    ACKNOWLEDGEMENTS

We thank Bert Herrera for tissue culture assistance and Di-hua He for technical support. We also appreciate the excellent editorial help given by Janet Wootten and manuscript preparation by Denise Christian.

    FOOTNOTES

This work was supported in part by the Medical Research Service of the Department of Veterans Affairs, by National Heart, Lung, and Blood Institute Grants HL-52136 and HL-58679, and by National Institute of Environmental Health Sciences Grant ES-06219.

The nucleotide sequence of the porcine calpain 3'-end fragment reported in this article has been deposited in the GenBank database (accession no. SSU71320).

Address for reprint requests: J. M. Patel, Research Service 151, VA Medical Center, 1601 SW Archer Rd., Gainesville, FL 32608-1197.

Received 31 December 1997; accepted in final form 11 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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