Mesenchymal stem cell contact promotes CCN1 splicing and transcription in myeloma cells

Abstract

CCN family member 1 (CCN1), also known as cysteine-rich angiogenic inducer 61 (CYR61), belongs to the extracellular matrix-associated CCN protein family. The diverse functions of these proteins include regulation of cell migration, adhesion, proliferation, differentiation and survival/apoptosis, induction of angiogenesis and cellular senescence. Their functions are partly overlapping, largely non-redundant, cell-type specific, and depend on the local microenvironment. To elucidate the role of CCN1 in the crosstalk between stromal cells and myeloma cells, we performed co-culture experiments with primary mesenchymal stem cells (MSC) and the interleukin-6 (IL-6)-dependent myeloma cell line INA-6. Here we show that INA-6 cells display increased transcription and induction of splicing of intron-retaining CCN1 pre-mRNA when cultured in contact with MSC. Protein analyses confirmed that INA-6 cells co-cultured with MSC show increased levels of CCN1 protein consistent with the existence of a pre-mature stop codon in intron 1 that abolishes translation of unspliced mRNA. Addition of recombinant CCN1-Fc protein to INA-6 cells was also found to induce splicing of CCN1 pre-mRNA in a concentration-dependent manner. Only full length CCN1-Fc was able to induce mRNA splicing of all introns, whereas truncated recombinant isoforms lacking domain 4 failed to induce intron splicing. Blocking RGD-dependent integrins on INA-6 cells resulted in an inhibition of these splicing events. These findings expand knowledge on splicing of the proangiogenic, matricellular factor CCN1 in the tumor microenvironment. We propose that contact with MSC-derived CCN1 leads to splicing and enhanced transcription of CCN1 which further contributes to the translation of angiogenic factor CCN1 in myeloma cells, supporting tumor viability and myeloma bone disease.

Figure 1

Splicing of CCN1 pre-mRNA and protein expression is induced by MSC contact in INA-6 cells. INA-6 cells were either cultured alone (-) or in the presence of MSC (+). After 24 h of co-culture, INA-6 cells were separated from MSC by washing. (A) Splicing pattern of CCN1 pre-mRNA was investigated by semi-quantitative PCR with four different intron-spanning primers in five independent experiments. The primers are labeled according to their exon positions. INA-6 control cells (-) express the intron-retaining pre-mRNA whereas co-cultured INA-6 cells (+) show an increased expression of the intron-free transcript. Housekeeping gene EEF1A1 served as control. Different gels are indicated by dashed lines. (B) CCN1 protein expression in INA-6 cells. Western blot analysis of three independent experiments shows an increased protein expression of CCN1 in INA-6 cells after MSC contact. Re-blotting for β-actin was used to confirm equal loading of protein.

Figure 2

Exogenous stimulation by recombinant CCN1-Fc induces splicing of CCN1 pre-mRNA in INA-6 cells. INA-6 cells were incubated with varying concentrations of CCN1-Fc for 24 h. Semi-quantitative PCR was performed with primers for CCN1 4-5 and EEF1A1. Densitometric analysis was used to determine the amount of intron-retaining and intron-free pre-mRNA isoform. Bar graphs showing the percentage of intron-free isoform (intron-free mRNA compared to total (intron-free + intron retained) mRNA), which is enhanced by increased concentration of CCN1-Fc. EEF1A1 was used to normalize data.

Figure 3

Domain 4 of CCN1-Fc is required to induce CCN1 pre-mRNA splicing. (A) INA-6 cells were incubated with either 2.5 μg/ml recombinant CCN1-Fc full-length protein, or an equivalent amount of truncated CCN1 isoforms (CCN1-Fc 1-3, CCN1-Fc 1-2, and CCN1-Fc 1). Semi-quantitative PCR with intron-containing CCN1 primers showed that exposure to truncated CCN1 proteins comprising domains 1, 1-2, or 1-3 did not induce complete CCN1 mRNA splicing. (B) Splicing of CCN1 pre-mRNA induced by 2.5 μg/ml full-length CCN1-Fc was reversed by GRGDSP peptide (50 μg/ml in culture media) but not by addition of the control peptide GRADSP. One representative experiment out of three is shown.

Figure 4

CCN1-Fc enhances INA-6 cell viability in the absence of IL-6. INA-6 cells were cultured with serum-reduced media for 24 h before stimulation with (A) CCN1-Fc or (B) CCN1-Fc and IL-6. After 24 h, cell viability was measured by quantification of ATP. In the absence of IL-6, INA-6 cell viability was significantly enhanced when stimulated with 0.1 μg/ml – 1 μg/ml CCN1-Fc, whereas incubation with 0.05 μg/ml – 2.5 μg/ml CCN1-Fc led to a slight enhancement of INA-6 cell viability in presence of IL-6. Results depicted graphically represent the mean + SD for four independent experiments. *p < 0.05 relative to control.