Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: A potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency

Abstract

In alpha1-AT deficiency, a misfolded but functionally active mutant alpha1-ATZ (alpha1-ATZ) molecule is retained in the endoplasmic reticulum of liver cells rather than secreted into the blood and body fluids. Emphysema is thought to be caused by the lack of circulating alpha1-AT to inhibit neutrophil elastase in the lung. Liver injury is thought to be caused by the hepatotoxic effects of the retained alpha1-ATZ. In this study, we show that several "chemical chaperones," which have been shown to reverse the cellular mislocalization or misfolding of other mutant plasma membrane, nuclear, and cytoplasmic proteins, mediate increased secretion of alpha1-ATZ. In particular, 4-phenylbutyric acid (PBA) mediated a marked increase in secretion of functionally active alpha1-ATZ in a model cell culture system. Moreover, oral administration of PBA was well tolerated by PiZ mice (transgenic for the human alpha1-ATZ gene) and consistently mediated an increase in blood levels of human alpha1-AT reaching 20-50% of the levels present in PiM mice and normal humans. Because clinical studies have suggested that only partial correction is needed for prevention of both liver and lung injury in alpha1-AT deficiency and PBA has been used safely in humans, it constitutes an excellent candidate for chemoprophylaxis of target organ injury in alpha1-AT deficiency.

Figure 1

Effect of chemical chaperones on secretion of α1-ATZ. CJZ12B cells (7, 8) were preincubated in control medium or medium supplemented with the designated drug as described in Materials and Methods. Cells were then subjected to pulse-chase radiolabelling, and the results were analyzed by immunoprecipitation followed by SDS/PAGE/fluorography. Molecular mass markers (in kDa) are shown on the right. The relative electrophoretic migration of the 55-kDa α1-ATZ polypeptide in the EC is indicated on the right by a triangular arrowhead. (A) CJZ12B cells incubated for 12 h in the absence or presence of 5 or 10% glycerol. (B) CJZ12B cells incubated for 12 h in the absence or presence of 150 mM TMAO. (C) CJZ12B cells incubated for 12 h in the absence or presence of 10 mM PBA. (D) Summary table of results from treatment of CJZ12B cells with several chemical chaperones. The data are expressed as percent α1-ATZ delivered to the EC after a 4-h chase, compared with the total α1-ATZ newly synthesized at t = 0 IC, as determined by densitometric analysis of gels. In each case, the pulse-chase experiment from the control and experimental conditions were exposed on the same film and a similar amount of α1-ATZ was present at t = 0 IC in the control and experimental conditions. Values represent the means ± 1.0 SD for the percent of α1-ATZ delivered to EC from three independent experiments in each case. The range of values for the control was 2.0–3.9%; glycerol, 23.0–27.2%; PBA, 15.0–18.4%; TMAO, 2.0–4.0%; D₂0, 2.0–4.0%; and betaine, 1.9–3.9%. Results were similar when phosphorimaging analysis was used to quantify results in two separate experiments for glycerol and PBA (data not shown).

Figure 2

Effect of PBA on secretion of α1-ATZ in a murine hepatoma cell line. Hepa1–6N2Z4 cells were preincubated for 12 h at 37°C in control medium in the absence (control) or presence of 10 mM PBA and then subjected to pulse-chase radiolabeling and gel analysis as described in the legend of Fig. 1. Molecular mass markers (in kDa) are shown at the right. The relative electrophoretic migration of the 55-kDa α1-ATZ polypeptide in the EC is indicated on the right by a triangular arrowhead.

Figure 3

Other effects of PBA on α1-AT. (A) Effect of PBA on synthesis of endogenous wild-type α1-AT in the human hepatoma cell line HepG2. HepG2 cells were preincubated for 12 h at 37°C in control medium or medium supplemented with 10 mM PBA. The cells were then pulse labeled for 20 min and lysed, and the cell lysates analyzed by immunoprecipitation followed by SDS/PAGE/fluorography. (B) Functional activity of mutant α1-ATZ secreted by CJZ12B (Left) and wild-type α1-AT secreted by HepG2 (Right) cells after treatment with PBA. Cells were subjected to a pulse-chase experiment and EC was harvested after a chase period of 4 h. Aliquots of the EC were incubated for 30 min at 37°C in the absence or presence of purified human neutrophil elastase in increasing concentrations as shown at the bottom. The reaction was terminated by the addition of PMSF to 2 mM, and the reaction mixtures subject to immunoprecipitation and SDS/PAGE/fluorography. The relative migration of the 55-kDa native α1-ATZ in the EC was indicated by the arrowhead in the left margin. The ≈75-kDa α1-AT–elastase complex is indicated by an asterisk in the right margin.

Figure 4

Effect of PBA on human α1-AT levels in PiZ mice. Seven PiZ mice were separated into two groups for administration of PBA or vehicle by gavage in a crossover design as described in Materials and Methods. (A) This graph shows the combined results for PBA in the two trial periods, including all seven PiZ mice at days 3 and 5 (while receiving PBA) and day 12 (off PBA for 7 days). The results are reported as means ± 1.0 SD for the increase in human α1-AT levels (μg/ml) compared with the baseline (day 0). The increases in blood levels of human α1-AT during PBA treatment on days 3 and 5 were significantly different from that on day 12 at P < .001 as determined by the Student's t test. There was no change in human α1-AT levels during administration of vehicle in any of the mice during either trial period (data not shown). (B) This graph shows the absolute levels of human α1-AT in four mice with baseline levels of 600–700 μg/ml. (C) This graph shows the absolute levels of human α1-AT in three PiZ mice with lower baseline levels (300–400 μg/ml).

Figure 5

Effect of temperature on the fate of α1-ATZ in CJZ12B cells. Cells were incubated for 12 h at normal growth temperature (37°C) or adjusted temperatures (27 or 42°C) and subjected to pulse-chase radiolabelling exactly as described in Materials and Methods. The relative migration of the 55-kDa α1-ATZ polypeptide in the EC is indicated by the arrowhead at the right margin. A 65-kDa polypeptide which coprecipitates with α1-ATZ in cell lysates at 42°C is indicated by an asterisk in the left margin.