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CADM1 isoforms differentially regulate human mast cell survival and homotypic adhesion.

Moiseeva EP, Leyland ML, Bradding P - Cell. Mol. Life Sci. (2012)

Bottom Line: CADM1 downregulation resulted in reduced viability and decreased expression of the pro-survival protein Mcl-1(L), but not Blc-2 or Bcl-X(L), and increased caspase-3/7 activity in both HMC-1 cells and HLMCs.This coincided with decreased basal Kit levels in HLMCs.The most highly expressed SP4 isoform is likely to contribute to MC aggregation and longevity in mastocytosis, and augment the pathophysiology of allergic diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection, Immunity and Inflammation, Institute for Lung Health, Clinical Sciences Wing, Glenfield Hospital, University of Leicester, Leicester LE3 9QP, UK. em9@le.ac.uk

ABSTRACT
Cell adhesion molecule 1 (CADM1), expressed by human lung mast cells (HLMCs), mediates their adhesion to airway smooth muscle (ASM), and contributes to ASM-dependent HLMC proliferation and survival. CADM1 is expressed in alternatively spliced isoforms, but those present in HLMCs and their function are not known. We cloned three functional and one cryptic non-functional isoform with alternative splicing between exons 7/11 and 1/2, respectively, from HLMCs and human MC lines (HMC-1 and LAD2). Differentiated HLMCs and LAD2 cells expressed the functional isoform SP4 containing exons 7/8/11 (~80% of clones), as well as SP1 (exons 7/8/9/11) and a novel SP6 (exons 7/8/9/10/11). In contrast, immature HMC-1 cells expressed only functional SP4. SP4 overexpression in HMC-1 cells and HLMCs augmented homotypic adhesion to a greater extent than SP1 in various conditions. In contrast, CADM1 downregulation abolished homotypic adhesion, indicating that CADM1 is the sole receptor mediating mast cell aggregation. CADM1-mediated adhesion was enhanced by the presence of cell survival factors. SP1 overexpression in HMC-1 cells compromised survival compared to SP4 overexpression or control. CADM1 downregulation resulted in reduced viability and decreased expression of the pro-survival protein Mcl-1(L), but not Blc-2 or Bcl-X(L), and increased caspase-3/7 activity in both HMC-1 cells and HLMCs. This coincided with decreased basal Kit levels in HLMCs. In summary, human MCs express multiple CADM1 isoforms which exhibit differential regulation of survival and homotypic adhesion. The most highly expressed SP4 isoform is likely to contribute to MC aggregation and longevity in mastocytosis, and augment the pathophysiology of allergic diseases.

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Modulation of CADM1 affects cell viability in HMC-1 cells. Transduced cells were washed and then incubated in IMDM alone for 48 h. The percentage of viable cells and caspase 3/7 activity (a) were measured in two experiments each performed in quadruplicate. Survival is shown as a percentage of cells surviving from the beginning of the experiment. **P < 0.01, ***P < 0.001. b Transduced HMC-1 cells were washed and incubated in IMDM with 1 μM A23187 for 24 h. Survival and caspase-3/7 activity were measured as in (a). Two experiments each performed in quadruplicate. **P < 0.01, ***P < 0.001 versus SP4. c Western blot of transduced cells at 0 and 44 h following removal of viruses in IMDM alone (representative of two to four experiments for different groups 30 μg/lane). Abs are shown on the right. d Protein bands, shown in (c) at a baseline (0 h) were quantified (n = 2). Bands in non-transduced, SP4 and SP1 groups, and non-transduced cells were expressed as percentages of GFP group; bands in Sh5 and Shm groups were expressed as percentages of LucSh group. Data for Bcl-2 and Bcl-XL are not shown. The data with 14 points for each protein (two experiments with seven groups each) were analysed for correlation. Regression analysis of these data is shown (e)
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Fig5: Modulation of CADM1 affects cell viability in HMC-1 cells. Transduced cells were washed and then incubated in IMDM alone for 48 h. The percentage of viable cells and caspase 3/7 activity (a) were measured in two experiments each performed in quadruplicate. Survival is shown as a percentage of cells surviving from the beginning of the experiment. **P < 0.01, ***P < 0.001. b Transduced HMC-1 cells were washed and incubated in IMDM with 1 μM A23187 for 24 h. Survival and caspase-3/7 activity were measured as in (a). Two experiments each performed in quadruplicate. **P < 0.01, ***P < 0.001 versus SP4. c Western blot of transduced cells at 0 and 44 h following removal of viruses in IMDM alone (representative of two to four experiments for different groups 30 μg/lane). Abs are shown on the right. d Protein bands, shown in (c) at a baseline (0 h) were quantified (n = 2). Bands in non-transduced, SP4 and SP1 groups, and non-transduced cells were expressed as percentages of GFP group; bands in Sh5 and Shm groups were expressed as percentages of LucSh group. Data for Bcl-2 and Bcl-XL are not shown. The data with 14 points for each protein (two experiments with seven groups each) were analysed for correlation. Regression analysis of these data is shown (e)

Mentions: Adenoviruses containing SP4–GFP (see Fig. 5b) and GFP were used to optimise transduction conditions according to the manufacturer’s recommendations. Expression of CADM1–GFP or GFP in HMC-1 cells 6 days post transduction resulted in visible expression at variable levels in 70 ± 15 and 69 ± 8% of cells, respectively, as previously reported [30]. Similarly, transduction of various HLMCs with these viruses for 6 days produced stable and reproducible fluorescence. Transduction of HMC-1 cells and HLMCs with a multiplicity of infection of 50 IU/cell (17 × 3 IU/cell for mixed sh RNAs) for 6 days were used in all experiments, unless stated otherwise.


CADM1 isoforms differentially regulate human mast cell survival and homotypic adhesion.

Moiseeva EP, Leyland ML, Bradding P - Cell. Mol. Life Sci. (2012)

Modulation of CADM1 affects cell viability in HMC-1 cells. Transduced cells were washed and then incubated in IMDM alone for 48 h. The percentage of viable cells and caspase 3/7 activity (a) were measured in two experiments each performed in quadruplicate. Survival is shown as a percentage of cells surviving from the beginning of the experiment. **P < 0.01, ***P < 0.001. b Transduced HMC-1 cells were washed and incubated in IMDM with 1 μM A23187 for 24 h. Survival and caspase-3/7 activity were measured as in (a). Two experiments each performed in quadruplicate. **P < 0.01, ***P < 0.001 versus SP4. c Western blot of transduced cells at 0 and 44 h following removal of viruses in IMDM alone (representative of two to four experiments for different groups 30 μg/lane). Abs are shown on the right. d Protein bands, shown in (c) at a baseline (0 h) were quantified (n = 2). Bands in non-transduced, SP4 and SP1 groups, and non-transduced cells were expressed as percentages of GFP group; bands in Sh5 and Shm groups were expressed as percentages of LucSh group. Data for Bcl-2 and Bcl-XL are not shown. The data with 14 points for each protein (two experiments with seven groups each) were analysed for correlation. Regression analysis of these data is shown (e)
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Related In: Results  -  Collection

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Fig5: Modulation of CADM1 affects cell viability in HMC-1 cells. Transduced cells were washed and then incubated in IMDM alone for 48 h. The percentage of viable cells and caspase 3/7 activity (a) were measured in two experiments each performed in quadruplicate. Survival is shown as a percentage of cells surviving from the beginning of the experiment. **P < 0.01, ***P < 0.001. b Transduced HMC-1 cells were washed and incubated in IMDM with 1 μM A23187 for 24 h. Survival and caspase-3/7 activity were measured as in (a). Two experiments each performed in quadruplicate. **P < 0.01, ***P < 0.001 versus SP4. c Western blot of transduced cells at 0 and 44 h following removal of viruses in IMDM alone (representative of two to four experiments for different groups 30 μg/lane). Abs are shown on the right. d Protein bands, shown in (c) at a baseline (0 h) were quantified (n = 2). Bands in non-transduced, SP4 and SP1 groups, and non-transduced cells were expressed as percentages of GFP group; bands in Sh5 and Shm groups were expressed as percentages of LucSh group. Data for Bcl-2 and Bcl-XL are not shown. The data with 14 points for each protein (two experiments with seven groups each) were analysed for correlation. Regression analysis of these data is shown (e)
Mentions: Adenoviruses containing SP4–GFP (see Fig. 5b) and GFP were used to optimise transduction conditions according to the manufacturer’s recommendations. Expression of CADM1–GFP or GFP in HMC-1 cells 6 days post transduction resulted in visible expression at variable levels in 70 ± 15 and 69 ± 8% of cells, respectively, as previously reported [30]. Similarly, transduction of various HLMCs with these viruses for 6 days produced stable and reproducible fluorescence. Transduction of HMC-1 cells and HLMCs with a multiplicity of infection of 50 IU/cell (17 × 3 IU/cell for mixed sh RNAs) for 6 days were used in all experiments, unless stated otherwise.

Bottom Line: CADM1 downregulation resulted in reduced viability and decreased expression of the pro-survival protein Mcl-1(L), but not Blc-2 or Bcl-X(L), and increased caspase-3/7 activity in both HMC-1 cells and HLMCs.This coincided with decreased basal Kit levels in HLMCs.The most highly expressed SP4 isoform is likely to contribute to MC aggregation and longevity in mastocytosis, and augment the pathophysiology of allergic diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection, Immunity and Inflammation, Institute for Lung Health, Clinical Sciences Wing, Glenfield Hospital, University of Leicester, Leicester LE3 9QP, UK. em9@le.ac.uk

ABSTRACT
Cell adhesion molecule 1 (CADM1), expressed by human lung mast cells (HLMCs), mediates their adhesion to airway smooth muscle (ASM), and contributes to ASM-dependent HLMC proliferation and survival. CADM1 is expressed in alternatively spliced isoforms, but those present in HLMCs and their function are not known. We cloned three functional and one cryptic non-functional isoform with alternative splicing between exons 7/11 and 1/2, respectively, from HLMCs and human MC lines (HMC-1 and LAD2). Differentiated HLMCs and LAD2 cells expressed the functional isoform SP4 containing exons 7/8/11 (~80% of clones), as well as SP1 (exons 7/8/9/11) and a novel SP6 (exons 7/8/9/10/11). In contrast, immature HMC-1 cells expressed only functional SP4. SP4 overexpression in HMC-1 cells and HLMCs augmented homotypic adhesion to a greater extent than SP1 in various conditions. In contrast, CADM1 downregulation abolished homotypic adhesion, indicating that CADM1 is the sole receptor mediating mast cell aggregation. CADM1-mediated adhesion was enhanced by the presence of cell survival factors. SP1 overexpression in HMC-1 cells compromised survival compared to SP4 overexpression or control. CADM1 downregulation resulted in reduced viability and decreased expression of the pro-survival protein Mcl-1(L), but not Blc-2 or Bcl-X(L), and increased caspase-3/7 activity in both HMC-1 cells and HLMCs. This coincided with decreased basal Kit levels in HLMCs. In summary, human MCs express multiple CADM1 isoforms which exhibit differential regulation of survival and homotypic adhesion. The most highly expressed SP4 isoform is likely to contribute to MC aggregation and longevity in mastocytosis, and augment the pathophysiology of allergic diseases.

Show MeSH
Related in: MedlinePlus