In the classical form of alpha1-antitrypsin (AT) deficiency, a point mutation in AT alters the folding of a liver-derived secretory glycoprotein and renders it aggregation-prone. In addition to decreased serum concentrations of AT, the disorder is characterized by accumulation of the mutant alpha1-antitrypsin Z (ATZ) variant inside cells, causing hepatic fibrosis and/or carcinogenesis by a gain-of-toxic function mechanism. The proteasomal and autophagic pathways are known to mediate degradation of ATZ. Here we show that the autophagy-enhancing drug carbamazepine (CBZ) decreased the hepatic load of ATZ and hepatic fibrosis in a mouse model of AT deficiency-associated liver disease. These results provide a basis for testing CBZ, which has an extensive clinical safety profile, in patients with AT deficiency and also provide a proof of principle for therapeutic use of autophagy enhancers.

Extensive documentation has validated the role of UV irradiation as a tumor initiator and promoter, inducing both squamous and basal cell carcinomas. Human epidermis is a tissue which undergoes active metabolism of arachidonic acid to prostaglandins which is regulated by the action of prostaglandin H synthase (also known as cyclooxygenase). One mechanism for the promotional activity of UV light may involve its ability to induce prostaglandin formation. Work in our laboratory has demonstrated that acute exposure of human keratinocytes to UVB irradiation results in increased production of prostaglandin E2 (PGE2). When cultured human keratinocytes were examined after irradiation with 30 mJ/cm2 UVB in vitro, Western blot analysis showed a 6-fold increase in COX-2 protein which was evident at 6 h and peaked 24 h after irradiation. Furthermore, when human subjects were irradiated on sun-protected skin with up to four times their minimal erythema dosage (MED) and biopsied 24 h later, upregulation of COX-2 protein expression was observed via immunofluorescence microscopy. RNAase protection assays supported this observation, showing induction of COX-2 message which peaked at approximately 12 h following irradiation in vitro. Furthermore, human squamous cell carcinoma biopsies exhibited strongly enhanced staining for COX-2 protein via immunohistochemistry and Western analysis when compared to normal non-sun-exposed control skin. Together, these data demonstrate acute upregulation of COX-2 via UVB irradiation and suggest the need for further studies of COX-2 expression as a potential pharmacological target mediating human skin tumor development.

In ␣ 1 -antitrypsin (␣1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein ␣1AT, ␣1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the "susceptible" subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant ␣1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant ␣1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant ␣1AT. The results show that ER retention of polymerogenic mutant ␣1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of ␣1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic ␣1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant ␣1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic ␣1AT mutants.In the classical form of ␣1AT 2 deficiency, the mutant ␣1ATZ molecule is retained in the endoplasmic reticulum (ER) of liver cells rather than secreted. There is an 85-90% reduction in ␣1AT levels in the blood and body fluids. This deficiency affects ϳ1 in 1800 live births and results in the premature development of pulmonary emphysema in adult life. Chronic liver disease develops in a subgroup of homozygotes, usually becoming evident during childhood. There is also an increased incidence of hepatocellular carcinoma later in life. Emphysema is caused by a loss-of-function mechanism whereby lack of ␣1AT in the lung allows proteolytic destruction of the connective tissue matrix. In contrast, liver injury appears to involve a gain-of-toxic-function mechanism whereby the accumulation of mutant ␣1ATZ in the ER damages liver cells (1).Nevertheless, relatively little is known about the factors that predispose the "susceptible" subpopulation of PIZZ individuals to liver disease and/or protect the remainder of the PIZZ population from liver disease. By using skin fibroblast cell lines from PIZZ individuals with or without liver disease engineered for expression of ␣1ATZ, we have shown previously that there is a lag in ER degradation of mutant ␣1ATZ in cells from PIZZ individuals with liver disease (2). These results provided evidence that the response of cells to the accumulation of this mutant protein in the ER, particularly the degradative machinery, could play a role in determining the susceptibility to liver disease among ...

Because retention of mutant ␣ 1 -antitrypsin (␣ 1 -AT) Z in the endoplasmic reticulum (ER) is associated with liver disease in ␣ 1 -AT-deficient individuals, the mechanism by which this aggregated glycoprotein is degraded has received considerable attention. In previous studies using stable transfected human fibroblast cell lines and a cell-free microsomal translocation system, we found evidence for involvement of the proteasome in degradation of Chem. 275, 25015-25022) found that degradation of ␣ 1 -ATZ in a stable transfected murine hepatoma cell line was inhibited by tyrosine phosphatase inhibitors, but not by the proteasomal inhibitor lactacystin and concluded that the proteasome was only involved in ER degradation of ␣ 1 -ATZ in nonhepatocytic cell types or in cell types with levels of ␣ 1 -AT expression that are substantial lower than that which occurs in hepatocytes. To examine this important issue in further detail, in this study we established rat and murine hepatoma cell lines with constitutive and inducible expression of ␣ 1 -ATZ. In each of these cell lines degradation of ␣ 1 -ATZ was inhibited by lactacystin, MG132, epoxomicin, and clasto-lactacystin ␤-lactone. Using the inducible expression system to regulate the relative level of ␣ 1 -ATZ expression, we found that lactacystin had a similar inhibitory effect on degradation of ␣ 1 -ATZ at high and low levels of ␣ 1 -AT expression. Although there is substantial evidence that other mechanisms contribute to ER degradation of ␣ 1 -ATZ, the data reported here indicate that the proteasome plays an important role in many cell types including hepatocytes.The classical and most common form of ␣ 1 -antitrypsin (␣ 1 -AT) 1 deficiency is a relatively unique genetic disease in that it is associated with injury to one tissue, pulmonary emphysema, by a loss-of-function mechanism and injury to another tissue, chronic hepatitis/hepatocellular carcinoma, by a gain-of-function mechanism. Many studies have provided evidence that emphysema results from lack of the anti-elastase activity of ␣ 1 -AT in the lung (reviewed in Refs. 1 and 2). Liver disease is due to toxic effects of aggregated ␣ 1 -ATZ retained in the ER of liver parenchymal cells. The gain-of-function mechanism is most clearly demonstrated by experiments in mice transgenic for human ␣ 1 -ATZ. These mice develop liver injury and hepatocellular carcinoma despite the fact that they have their own endogenous anti-elastases (3-5).The mutant Z allele of ␣ 1 -AT is characterized by a single nucleotide substitution, which results in the replacement of glutamate 342 by a bulky lysine residue (1, 2). The studies of Carrell and Lomas (6, 7) have shown that this substitution renders the ␣ 1 -AT molecule more susceptible to polymerization and that highly ordered aggregates accumulate in the ER of liver cells.One interesting observation, arising from unbiased nationwide screening studies of ␣ 1 -AT deficiency in Sweden, indicates that only 10 -15% of deficient individuals develop clinically significant liver disease (8,9...