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Extranuclear sequestration of phospho-Jun N-terminal kinase and distorted villi produced by activated Rac1 in the intestinal epithelium of chimeric mice

¹ Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA
² Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA

View larger version (88K): [in a new window]   Fig. 1. Increased phosphorylation of 46 kDa Jnk in the apical cytoplasmic compartment of E18.5 129/Sv-Rac1Leu61 intestinal epithelium. (A) Replicate immunoblots of small intestinal extracts (50 µg protein/lane) from E18.5 Rac1Leu61 and normal chimeric mice were probed with antibodies that recognize Rac1, p-Jnk, the 46 kDa or 55 kDa forms of Jnk (designated Jnk-Ab1 and Jnk-Ab2, respectively), and p-c-Jun. (B-S) Sections of E18.5 jejunum from Rac1Leu61 (B-I; L-S) and normal (J,K) chimeric mice. Each antibody staining (C,E,G,I,K,M,O,Q,S) is paired with X-Gal staining (B,D,F,H,J,L,N,P,R) of the same section to show the epithelial cell genotype. At E18.5, the proliferative zone lies in the intervillus region (below broken line) positioned between rudimentary villi (above broken line). Sections from Rac1Leu61 (C,E) and normal chimeric jejunums (K) were stained with rabbit anti-p-Jnk, Cy3-tagged sheep anti-mouse Ig (red) and bis-benzimide (dark blue). 129/Sv epithelial cells in the Rac1Leu61 chimera show markedly enhanced p-Jnk staining localized principally to the apical cytoplasm (arrows). (G) High-power view of 129/Sv villus epithelial cells from a Rac1Leu61 chimera, stained with rabbit anti-Rac1, donkey anti-rabbit Ig and bis-benzamide. Although most of the immunoreactive protein is cytoplasmic, a small fraction localizes to the apical cell surface (arrow). (I) Section from a Rac1Leu61 chimera stained with the same reagents as in C,E, except that the p- Jnk mAb was pre-incubated with phosphorylated Jnk peptide. (M) Section stained with Jnk-Ab1 that recognizes total cellular 46 kDa Jnk, Cy3-tagged donkey anti-rabbit Ig, and bis-benzimide. Jnk (red) localizes to the apical cytoplasm, cell-cell and cell-substrate borders of both 129/Sv-Rac1Leu61 and B6-ROSA26 villus and intervillus epithelial cells. (O) Section incubated with Jnk-Ab2 that recognizes total cellular 55 kDa Jnk, Cy3-labeled donkey anti-rabbit Ig and bis-benzimide. 55 kDa Jnk localizes to the cytoplasm and nuclei of both 129/Sv-Rac1Leu61 and B6-ROSA26 villus and intervillus epithelial cells. (Q) Section stained with mouse anti-p-c-Jun, Cy3-sheep anti-mouse Ig and bis-benzimide. The nuclear staining for p-c-Jun is not appreciably different between the 129/Sv-Rac1Leu61 and B6-ROSA26 epithelial populations (compare B6-ROSA26 cells indicated by arrows to 129/Sv-Rac1Leu61cells indicated by a box). (S) Section from a Rac1Leu61 chimera stained with the same reagents as in Q, except that the p-c-Jun mAb was pre-incubated with phosphorylated c-Jun peptide. Scale bars: B,C,H-S, 25 µm; D-G, 10 µm.

View larger version (78K): [in a new window]   Fig. 2. Rac1Leu61 does not activate other MAP kinases and is associated with Pak1 redistribution in E18.5 mice. (A) Replicate immunoblots of small intestinal extracts from E18.5 Rac1Leu61 and normal chimeric mice (lanes 1 and 2, respectively) were probed with antibodies to p-Erk, p-p38, Pak1, and p215Tyr-c-Src. (B,C) Section from a Rac1Leu61 chimera stained with X-Gal (B) and with mouse anti-p-Erk, Cy3-tagged sheep anti-mouse Ig, plus bis-benzimide (C). There is no detectable difference in nuclear and cytoplasmic staining between neighboring 129/Sv-Rac1Leu61 and B6-ROSA26 epithelial cells. (D-G) Sections from Rac1Leu61 (D,E) and normal (F,G) chimeric jejunum stained with X-Gal and rabbit anti-Pak1, Cy3-donkey anti-rabbit Ig, plus bis-benzimide. E shows that Pak1 staining is more prominent in the apical cytoplasm of 129/Sv-Rac1Leu61 intervillus and villus epithelial cells when, compared with normal adjacent B6-ROSA26 cells (see inset; arrowhead). G shows that Pak1 is diffusely distributed in the cytoplasm of epithelial cells of both genotypes in normal chimeras. (H,I) Intervillus region from a Rac1Leu61 mouse stained with X-Gal and rabbit anti-p215 Tyr-c-Src, Cy3-donkey anti-rabbit Ig, plus bis-benzimide. Rac1Leu61 has no apparent effect on c-Src localization to cell-cell and cell-substrate interfaces. Scale bars: 25 µm.

View larger version (69K): [in a new window]   Fig. 3. Rac1Leu61 is associated with markedly widened villi in adult chimeric mice (A) Idealized three-dimensional drawing of the mouse small intestine from a normal C57Bl/6-ROSA26129/Sv mouse. The front of the intestine has been 'sectioned’ to show the relationship of individual crypts and adjacent villi. In the adult chimeras, crypts are ‘monoclonal’: composed of either B6-ROSA26 or 129/Sv epithelial cells, but not a mixture of both. Some villi are ‘polyclonal’: supplied by crypts of both genotype. (B) Whole-mount preparation of jejunum from a 9-month-old normal chimeric mouse. The intestine has been opened, fixed and stained with X-Gal. The white epithelium is 129/Sv. The blue epithelium is composed of B6-ROSA26 cells that express lacZ. (C) 9-month-old Rac1Leu61chimeric jejunum prepared as in B. Compared with the normal 129/Sv villi in B, 129/Sv-Rac1Leu61 villi are widened, in some cases coalescing to form very unusual, undulating, slab-like structures (arrowheads). The arrows point to sharply demarcated stripes of blue cells that emanate from B6-ROSA26 crypts supplying two widened polyclonal villi. The inset shows the results of a RT-PCR analysis of normal and Rac1Leu61 RNAs. The 250bp PCR product is from the Fabpl-Rac1Leu61 transcript. Scale bars: 235 µm.

View larger version (79K): [in a new window]   Fig. 4. 129/Sv-Rac1Leu61 crypts in adult chimeric mice are elongated and show increased proliferation. (A-C) Sections from an adult Rac1Leu61 chimeric jejunum. (A) X-Gal genotyping of 129/Sv and B6-ROSA26 epithelial cells. The broken line indicates the location of crypt-villus junctions. (B) Same section as in A stained with mouse anti-c-Myc, Cy3-labeled sheep anti-mouse Ig and bis-benzimide. Cytoplasmic c-Myc staining (red) is more prominent in the 129/Sv villus epithelium (arrowhead) compared with crypt (arrows) epithelium. (C) High-power view of the 129/Sv and adjacent B6 crypts noted in B. The arrowhead highlights the modest increase in c-Myc staining in the upper part of the 129/Sv-Rac1Leu61 crypt compared with the neighboring B6-ROSA26 crypt. (D,E) Section from a normal 9-month-old chimera. (D) X-Gal genotyping. (E) Section stained with the same reagents as in B. Asterisks in B,E indicate Cy3-positive lymphocytes in the lamina propria that react with the mouse secondary antibody. This lamina propria staining can be used as a reference internal control: even with the increased exposure in E, no epithelial c-Myc staining is detectable in the normal chimera. (F,G) X-Gal plus Hematoxylin and Eosin (F), or nuclear Fast Red (G) stained sections. The arrow in F denotes a 129/Sv crypt that appears elongated when compared to a neighboring B6-ROSA26 crypt (arrowhead). In G, arrowheads point to some of the increased M-phase cells present in a 129/Sv crypt. (H) Quantitative morphometric studies reveal a 2.5-fold increase in mitotic index in 129/Sv-Rac1Leu61 crypts. See text for definition of the mitotic index. Scale bars: 25 µm.

View larger version (76K): [in a new window]   Fig. 5. The effect of Rac1Leu61 on the intracellular epithelial distribution of ß-actin and p-Jnk in adult mice. (A) Section containing neighboring 129/Sv and B6-ROSA26 jejunal villi from a 9-month-old Rac1Leu61 chimeric mouse, stained with FITC-labeled mouse anti-ß-actin and bis-benzimide. The apical cytoplasmic staining in the 129/Sv-Rac1Leu61villus epithelium (arrow) is diminished relative to the adjacent B6-ROSA26 epithelium (arrowhead). (B) Polyclonal villus from a normal chimera stained with the same reagents as in A. Actin staining is equivalent in 129/Sv and B6-ROSA26 epithelium. (C) Polyclonal villus from an adult chimeric-transgenic mouse stained with mouse anti-p-Jnk, Cy3-labeled sheep anti-mouse Ig and bis-benzimide. The p-Jnk staining is increased in the apical cytoplasm of 129/Sv-Rac1Leu61 villus epithelial cells (arrow) compared to B6-ROSA26 cells (arrowhead). (D) Section from a normal chimeric jejunal villus stained with the same reagents as in C. Increased exposure of D (compare the intensity of staining of lamina propria lymphocytes in C) shows faint but equivalent p-Jnk staining in the cytoplasm of 129/Sv and B6-ROSA26 epithelial cells. (E,F) Pak1 (E) and Rac1 (F) distribution in adult villus epithelium. In E, villi have been sectioned perpendicular to the crypt-villus axis. Pak1 (red) is prominent in the apical cytoplasm of epithelial cells (arrow), irrespective of genotype (B6-ROSA26 cells are shown in this example). Rac1 (red in panel F) is evident throughout the cytoplasm. Scale bars: 25 µm.