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First published online March 29, 2004
doi: 10.1242/10.1242/jcs.01016

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Organ-specific stress induces mouse pancreatic keratin overexpression in association with NF-B activation

Bihui Zhong^1,2,3,*, Qin Zhou^1,2, Diana M. Toivola^1,2, Guo-Zhong Tao^1,2, Evelyn Z. Resurreccion^1,2 and M. Bishr Omary^1,2,

¹ VA Palo Alto Health Care System, Department of Medicine, 3801 Miranda Avenue, 154J, Palo Alto, CA 94304, USA
² Digestive Disease Center, Stanford University School of Medicine, Stanford, CA 94305, USA
³ Division of Gastroenterology, The First Affiliated Hospital of Sun Yet-sen University, Guangzhou 510080, China

View larger version (21K): [in a new window]   Fig. 1. Keratin content in mouse pancreas, liver and small intestine. (A) Liver, pancreas and small intestine were removed from Balb/c mice, and an enriched keratin fraction was obtained using high salt extraction (HSE) as described in Materials and Methods. Proteins were separated by SDS-PAGE, followed by Coomassie blue staining. Assignment of individual keratins was confirmed by two-dimensional gel analysis and immunoblotting using keratin-specific antibodies (not shown) (Zhou et al., 2003). (B) The indicated amounts (in µg) of serially diluted protein represent pancreatic keratins isolated by HSE or total pancreas lysates separated by SDS-PAGE. Resolved proteins were visualized by Coomassie staining or were analyzed by immunoblotting using keratin-specific antibodies. Similar analysis was done for keratins isolated from Balb/c mouse liver and distal ileum (not shown). (C) A summary of percent keratin protein contents in mouse liver, pancreas and small intestine (determined as described in Materials and Methods).

View larger version (72K): [in a new window]   Fig. 2. Histologic, K8/K18/K19/K20 protein and mRNA analysis in caerulein-induced pancreatitis. (A) Mice were injected with caerulein (50 µg/kg, ip) seven times at hourly intervals, and euthanized at different time points (1-240 hours), after which pancreata were fixed then stained with hematoxylin and eosin. All time-points are given as hour after the first injection, and control mice were euthanized 48 hours after vehicle-alone administration (7 saline injections). a=control; b=6 hours after injection of caerulein 6x hourly; c=48 hours after injection of caeruelin 7x hourly; and d=240 hours after injection of caeruelin 7x hourly. (B) After caerulein injections (see Materials and Methods) pancreata were collected at the indicated time-points (two mice (1, 2) per time-point) then homogenized. Protein homogenates were separated by SDS-PAGE (20 µg/lane) then blotted using antibodies to the indicated antigens. Control mice `C' were euthanized 48 hours after seven saline injections. (C) HSE or pancreatic tissue homogenates were prepared from mice injected with caerulein or saline (48 hours, two mice/condition). The amount of keratin isolated by HSE was normalized to the content of the detergent-soluble supernatant protein that was generated during the first step of the HSE procedure (see Materials and Methods), then analyzed by SDS-PAGE and densitometric scanning (to quantify fold-increase in keratins upon caerulein exposure). (D) Total RNA was isolated from pancreata 48 hours after caerulein or saline injection (two mice (M1, M2) per condition) followed by RT-PCR amplification of actin or the indicated keratin cDNAs. (E) Total pancreas RNA was isolated 48 hours after saline (2 mice) or caerulein (3 mice) treatment, or from the small intestine (SI, used as a control for K20 expression) of Balb/c mice (M1). The RNA was reverse transcribed using K20 primers (upper panel), followed by nested PCR using internal K20 primers (middle panel). HSE from the indicated tissues were also obtained and blotted using K20-specific Ab (lower panel). No sample was loaded in lane 6 in order to separate the small intestinal control from the remaining samples.

View larger version (58K): [in a new window]   Fig. 6. NF-B activity and keratin mRNA expression after ex vivo treatment of isolated acini with caerulein +/–MG-132. (A) Lanes 1-6: freshly isolated mouse pancreatic acini were treated with caerulein for 1-4 hours (37°C) followed by nuclear protein extraction and electrophoretic mobility shift assay (EMSA) as described in Materials and Methods. Binding in the presence of excess unlabeled NF-B-binding oligonucleotide (+probe, lane 6) is included as a specificity control. Lanes 7-12: acini were incubated in the presence or absence of caerulein for 2 hours (±MG-132 for 1 hour before the caerulein incubation) followed by nuclear protein extraction and EMSA as in lanes 1-6. (B) Pancreatic acini were incubated with caerulein (0.1 µM, 4 hours) with or without pretreatment with MG-132 (50 µM, 1 hour). Total RNA was isolated followed by real-time RT-PCR analysis of K8, K18 and K19 mRNA as described in Materials and Methods. Expression of each keratin mRNA was normalized to expression of the L7 gene (not shown). Results represent the mean±s.d. of three independent experiments. *P<0.01 vs. control; **P<0.01 vs. caerulein.

View larger version (126K): [in a new window]   Fig. 3. Keratin filament dynamics after caerulein-induced pancreatitis. Pancreata from control (saline-injected) and caerulein-injected (48 hours and 240 days) mice were processed for immunofluorescence staining with antibodies to K8/K18 (a-c), K19 (d-f) and K20 (g-i). Bar, 20 µm.

View larger version (58K): [in a new window]   Fig. 4. Keratin expression on CDD-induced pancreatitis. (A) Mice were fed a CDD for 3 days, followed by harvesting of pancreata from CDD-fed or from normal-diet-fed mice at various time-points then fixing and staining with hematoxylin and eosin. a=control diet; b=3 days of CDD feeding; c=3 days of CDD feeding then 1 day or normal diet; d=3 days of CDD feeding then 7 days or normal diet. (B) Pancreata were isolated from control-diet-fed (C) or CDD-fed mice. Feeding was done for 3 days followed by switching to a normal diet for 1 day (4d), 2 days (5d), 5 days (8d) or 7 days (10d). Pancreas fragments were solubilized in sample buffer, then equal amounts of protein (20 µg/lane) were analyzed (two separate mice/time-point) by blotting with antibodies to the indicated proteins.

View larger version (36K): [in a new window]   Fig. 5. Keratin expression after hyperthermia and water-immersion stresses. (A) Mouse core body temperature was increased to 42°C and maintained at this temperature for 20 minutes. Mice were then returned to their normal environment and allowed to maintain their usual activity and food/water intake. Pancreata were harvested from control non-heat exposed mice (C) or from the heat-stressed mice 0, 3, 6, 24 and 48 hours after the heat exposure. Two mice were used for each condition. Pancreatic total tissue homogenates were prepared and immunoblotted with antibodies to the indicated antigens. (B) Mice were immersed in a water bath (23°C) for 6 hours as described in Materials and Methods, then returned to their cages and allowed to maintain their usual activity and food/water intake. Pancreata were harvested from control non-water-immersed mice (C) or from the water-immersed mice 3, 6, 9 and 12 hours after removal from the water immersion. Total pancreatic protein extracts were prepared then analyzed by blotting as in A. Two mice were used for each condition.

View larger version (27K): [in a new window]   Fig. 7. Ex vivo acinar keratin mRNA expression after pretreatment with PDTC or Act-D. Isolated acini were incubated with medium±caerulein. The acini that were stimulated with caerulein were pre-incubated with 10, 25 or 50 µM of PDTC (A) or 0.5 or 2 µg/ml of Act-D (B) for 30 minutes followed by adding caerulein for 4 hours. The acinar cultures in B that did not include Act-D also included 0.1% DMSO (the solvent used for Act-D). Acini were then harvested and washed twice with PBS, followed by processing for real-time RT-PCR. L7 was used as internal control. The results represent mean±s.d. of three independent experiments. *P<0.01 vs. control; **P<0.01 vs. caerulein

View larger version (32K): [in a new window]   Fig. 8. Summary of generalized versus organ-specific stress on keratin expression. Generalized forms of stress, such as heat, which upregulates hsp70, do not have an impact on keratin expression levels in Balb/c mouse pancreas. By contrast, organ-specific stress that targets the pancreas, such as caerulein or CDD, increase pancreatic keratin levels without having a significant effect on hsp70/60 levels. The increased keratin expression in response to caerulein occurs in association with NF-B activation. Similar findings, in terms of organ-specific effects on keratin overexpression, have also been noted in the liver (Cadrin et al., 2000; Denk et al., 2000; Fickert et al., 2002). For example, feeding mice the hepatotoxins griseofulvin (GF) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) also results in keratin overexpression. However, not all forms of liver injury induce keratin overexpression, as feeding mice a lithogenic diet causes liver injury without affecting keratin expression levels. The lithogenic diet also results in cholelithiasis and gall bladder injury, with concurrent overexpression of gallbladder keratins (Tao et al., 2003).

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