CADM1 isoforms differentially regulate human mast cell survival and homotypic adhesion

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.

Fig. 1

CADM1 is highly expressed in HMC-1 cells compared to HLMCs. Surface CADM1 expression is shown as a plot of geometric means (a) (n = 3, **P < 0.01) and a representative histogram for HMC-1 cells and HLMCs from donors D552–D554 (b). c Western blot of protein expression in HMC-1 cells and HLMCs from donors D520, D528 and D529. d Relative CADM1 mRNA levels are shown as a ratio of CADM1 mRNA molecules to normalisers [10³ actin B (ACTB) mRNA and 10⁶ 18S rRNA] (n = 3, ***P < 0.001)

Fig. 2

Multiple CADM1 isoforms are present in human mast cells. a The diagram summarises CADM1 isoforms cloned from HLMCs and cell lines with novel isoforms, SP6 and c15, shown in grey. The exons present in each clone are depicted not to scale. Alternatively spliced exons are coloured grey. The percentages of each isoform present among clones are shown for HLMCs, D449 and D450, and cell lines, HMC-1 and LAD2. The estimated molecular weight of each isoform is shown in kDa on the right. The numbers of analysed clones are shown in italics. For modulation of CADM1 expression, HMC-1 cells were transduced with adenovirus carrying SP4, SP1, SP4–GFP or CADM1 sh RNAs (Shm indicates mixed Sh RNAs). Luc sh RNA and GFP adenoviruses were used as controls. Surface and total CADM1 protein were detected by FACS (b) (n = 4, **P < 0.01, ***P < 0.001 compared to no virus) or western blotting (c) (representative of two experiments), respectively

Fig. 3

Modulation of CADM1 expression markedly affects homotypic mast cell–cell adhesion. a HMC-1 cells (top panel representative of two experiments) and HLMCs (bottom panel representative of one experiment with two pooled samples), were transduced with adenovirus carrying SP4, SP1 or CADM1 sh RNAs for 6 and 4 days, respectively. Luc sh RNA and GFP adenoviruses were used as controls. Transduced cells were resuspended as single cell suspension and incubated in growth medium for 3 h at 37°C. Original magnification ×100; each experiment was performed in quadruplicate. The cross-sectional areas of the largest aggregates were estimated on photographs of four wells for each condition (b) (n = 40, **P < 0.01, ***P < 0.001)

Fig. 4

CADM1-mediated adhesion is influenced by CADM1 isoforms and survival signalling. a Transduced HMC-1 cells were cultured for 48 h in IMDM + 10% FCS (top panel) or in IMDM alone (bottom panel). Arrows indicate cells with membrane blebbing. Original magnification ×200; experiments performed in sextuplicate. The cross-sectional areas of the largest aggregates were estimated on photographs of four wells for each condition (b) (n = 40, *P < 0.05, **P < 0.01, ***P < 0.001). c HLMCs transduced with GFP and LucSh viruses (left panel representative of one donor, each assessed in quadruplicate) or SP4 and Sh5 adenoviruses (right panel representative of two donors, each assessed in quadruplicate) for 5 days, washed and grown for 72 h in 50% growth medium (50% HLMC medium/50% IMDM), or in IMDM alone. Original magnification ×100

Fig. 5

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)

Fig. 6

Modulation of CADM1 affects cell viability in HLMCs. a HLMC survival and caspase-3/7 activity were measured in transduced HLMCs after incubation for 72 h. All were measured in triplicate for each donor (n = 3). Survival in IMDM alone is shown as the percentage of cells present in 50% growth medium. Caspase 3/7 activity is shown in the presence (+) or absence (−) of growth factors. *P < 0.05, **P < 0.01, ***P < 0.001. b Pooled HLMCs from donors (D613 + D618) and (D616 + 619), transduced with SP4 or Sh5, were analysed by western blotting (25 μg/lane). Protein bands in (b) were quantified, normalised to non-transduced group and analysed for correlation (six points for each protein from two pools with three groups each) (c). d Western blot of proteins (25 μg/lane) from HLMCs D624, transduced with GFP and LucSh viruses

Fig. 7

A model of adhesion mediated by SP4 and SP1. CADM1 monomers, shown as grey molecules, are inactive in adhesion. When cells adhere, CADM1 forms dimers, shown as red molecules, which are actively involved in adhesion. a Two cells express only SP4, which forms active dimers involved in cell adhesion. b Two cells express both SP4 and SP1 (molecule with additional protein sequence encoded by exon 9, shown as a blue box in SP1 stalk near cell membrane) with the same density along cell membrane (orange line). Because SP4 and SP1 extracellular domains have different lengths, they cannot align properly and form dimers. The total number of CADM1 dimers, involved in cell adhesion, is reduced on the cell surface. However, over time, most of the CADM1 molecules would be able to find a matching partner to dimerise with and cells would be able to adhere as strongly as cells expressing only one CADM1 isoform