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J. Cell Sci. 119 (7): 1320-1328

Apical localization of ITPK1 enhances its ability to be a modifier gene product in a murine tracheal cell model of cystic fibrosis

Ling Yang1,2, Jeff Reece2, Sherif E. Gabriel3, and Stephen B. Shears1,2,*

1 Inositol Signaling Section, and N.I.E.H.S. / N.I.H. / D.H.S.S., Research Triangle Park, NC 27709, USA
2 Laboratory of Signal Transduction, N.I.E.H.S. / N.I.H. / D.H.S.S., Research Triangle Park, NC 27709, USA
3 Department of Pediatrics and Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA


Figure 1
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Fig. 1. Ins(3,4,5,6)P4 inhibits Ca2+-dependent Cl- secretion across WT and CF MTE monolayers. (A-D) ISC across WT (blue traces) and CF (red traces) MTE monolayers was measured as described in Materials and Methods. Incubation time was set to zero at the point at which UTP (100 µM) was added to the apical chamber. Where indicated, Bt2Ins(3,4,5,6)P4/PM and Bt2Ins(1,4,5,6)P4/PM were added to the apical chamber of the Ussing apparatus at a concentration of 50 µM, 2 hours prior to the addition of UTP. KN93 was added at a concentration of 5 µM, 2 hours prior to UTP addition. Representative traces are shown in A-D. (E) Graphs show the average height of {Delta}ISC peaks for the various experiments (mean ± s.e.m. of 4-6 determinations). Numbers above each bar indicate its percentage value relative to vehicle-treated cells within the same group [either WT (blue bars) or CF (red bars)].

 

Figure 2
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Fig. 2. Inositol-phosphate profile in MTEs. (A,B) Cells were labeled for 4 days with [3H]inositol. Cells were treated with either (A) vehicle or (B) 100 µM UTP for 90 seconds and the inositol phosphate levels were determined by HPLC as described in Materials and Methods. A representative experiment is shown.

 

Figure 3
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Fig. 3. PLC activity in MTEs activated with UTP. WT ({blacktriangleup}) and CF (bullet) cells were labeled for 4 days with [3H]inositol. Cells were treated with vehicle (the zero time point) or UTP (for 30, 60 or 90 seconds). Individual inositol phosphate levels were determined by HPLC as described in Materials and Methods. PLC activity is computed from the increase in InsP1 + InsP2 + InsP3 + InsP4 but excluding Ins(3,4,5,6)P4 (see Carew et al., 2000Go for an explanation). Data represent mean ± s.e.m. of five experiments.

 

Figure 4
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Fig. 4. Changes in levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4)P3 and Ins(3,4,5,6)P4 following purinergic activation of MTEs. WT ({blacktriangleup}) and CF (bullet) MTEs were labeled for 4 days with [3H]inositol. Cells were treated with vehicle (the zero time point) or UTP (for 30, 60 or 90 seconds). Levels of the individual inositol phosphate are as indicated. (A), Ins(1,4,5)P3; (B), Ins(1,3,4,5)P4; (C), Ins(1,3,4)P3; (D), Ins(3,4,5,6)P4. Data represent mean ± s.e.m. of five experiments. *P<0.03; **P<0.02.

 

Figure 5
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Fig. 5. Differences in ITPK1 expression in WT and CF MTEs and its impact upon synthesis of InsP5 and InsP6. (A-C) Quantitative PCR was used to assess the degree of ITPK1 expression in the two cell types, relative to the GAPDH control (A), as indicated in the Materials and Methods. Data represent mean ± s.e.m. of four experiments (P<0.002). Cellular levels of (B) InsP5 and (C) InsP6 (mean ± s.e.m. of five experiments) were determined as described in Fig. 2; levels of these two polyphosphates did not change following the addition of UTP (data not shown).

 

Figure 6
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Fig. 6. Analysis by confocal immunofluorescence microscopy of the decrease in ITPK1 expression by RNAi in MTEs. (A,B) WT MTEs grown on glass cover slips were transfected with either control, (A) non-targeting siRNA, or (B) ITPK1-specific siRNA as described in Materials and Methods. Both horizontal (X-Y) optical scanning from Z-stacks, and vertical (X-Z) scans are shown. Dotted lines show locations of accompanying perpendicular scans. ITPK1 was visualized with Alexa Fluor 488 (green) and nuclei with DAPI (blue). Bar, 10 µm. (C) Quantification of the intensity of the conjugated Alexa Fluor 488 throughout the stack volume, normalized to cell number. Data represent mean ± s.e.m. of ten independent experiments.

 

Figure 7
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Fig. 7. Analysis by confocal immunofluorescence microscopy of ITPK1 localization in polarized MTE monolayers. Cells were grown on membrane filters and prepared for immunofluorescence confocal microscopy as described in Materials and Methods. (A-G) Cells photographed in a horizontal (X-Y) optical section, with a corresponding vertical (X-Z) optical scans; (A-D) WT cells, (E-G) CF monolayers. Nuclei were stained with DAPI (A,E; blue), CFTR was stained with Alexa Fluor 546-labeled antibody (B; red) and ITPK1 was stained with Alexa Fluor 488-labeled antibody (C,F; green). The X-Z images of D and G include a white signal that is the confocal reflection from the filter. Bars, 10 µm.

 


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