Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells

Abstract

Junctional adhesion molecules (JAMs) are expressed in diverse types of stem and progenitor cells, but their physiological significance has yet to be established. Here, we report that JAMs exhibit a novel mode of interaction and biological activity in adipose-derived stromal/stem cells (ADSCs). Among the JAM family members, JAM-B and JAM-C were concentrated along the cell membranes of mouse ADSCs. JAM-C but not JAM-B was broadly distributed in the interstitial spaces of mouse adipose tissue. Interestingly, the JAM-C ectodomain was cleaved and secreted as a soluble form (sJAM-C) in vitro and in vivo, leading to deposition in the fat interstitial tissue. When ADSCs were grown in culture plates coated with sJAM-C, cell adhesion, cell proliferation and the expression of five mesenchymal stem cell markers, Cd44, Cd105, Cd140a, Cd166 and Sca-1, were significantly elevated. Moreover, immunoprecipitation assay showed that sJAM-C formed a complex with JAM-B. Using CRISPR/Cas9-based genome editing, we also demonstrated that sJAM-C was coupled with JAM-B to stimulate ADSC adhesion and maintenance. Together, these findings provide insight into the unique function of sJAM-C in ADSCs. We propose that JAMs contribute not only to cell-cell adhesion, but also to cell-matrix adhesion, by excising their ectodomain and functioning as a niche-like microenvironment for stem and progenitor cells.

Keywords: junctional adhesion molecule; mesenchymal stem cell; niche; shedding; stem cell; tight junction.

Figure 1

JAM-B and JAM-C are concentrated on cell membranes of mouse adipose-derived stromal/stem cells (ADSCs). RT-PCR (A) and Western blot (B) for the indicated molecules in ADSCs derived from three mice (#1–3). Mouse kidney (for Jam-4) and spleen tissues (for other junctional adhesion molecules; JAMs) are used as positive controls (Ctrl). P, passage. (C) Confocal images of ADSCs stained for the indicated markers. Arrowheads show JAM-B- and JAM-C-immunoreactive signals on the cell membranes of ADSCs. Squares indicate the enlarged areas. Scale bar, 50 µm.

Figure 2

Fluorescence-activated cell sorting (FACS) profiles of the stromal vascular fraction (SVF) of the mouse adipose tissue. (A) Association between the expression of mesenchymal stem cell (MSC) markers CD44, CD105, CD140a and Sca-1 and JAM-B or JAM-C expression. (B) The JAM-C expression in the Sca1 /CD31⁻/CD45⁻/Ter119⁻ cell lineage.

Figure 3

JAM-C is widely distributed in the fat interstitial tissues. (A–C) Confocal images of mouse ADSCs stained for the indicated markers. Asterisks indicate lipid droplets in differentiated adipocytes, and arrowheads show colocalization of JAM-C and ether type III collagen or heparan sulfate proteoglycan (HSPG). Squares show the enlarged areas. Scale bars, 50 µm.

Figure 4

The cleaved JAM-C ectodomain is accumulated in the fat interstitial tissues. (A) Schematic illustration for detecting full-length JAM-C (fJAM-C) and/or soluble JAM-C (sJAM-C) using JAM-C (N) and JAM-C (C) antibodies (Abs) targeting the N- and C-termini, respectively. (B) Knockdown of the Jam3 gene encoding mouse JAM-C in ADSCs using the CRISPR method. (C) Western blot for the indicated proteins in the whole-cell lysates and supernatants of the revealed ADSCs. (D) Western blot for the indicated proteins in the whole-cell lysates of mouse adult spleen tissue (control), adipose tissue and ADSCs. (E) Confocal images of mouse adipose tissue stained with JAM-C (N) and JAM-C (C) Abs. Asterisks indicate lipid droplets in differentiated adipocytes. Squares show the enlarged areas. Scale bar, 50 µm.

Figure 5

Soluble JAM-C functions as the niche for ADSCs. (A) Schematic illustration of recombinant JAM-B (rJAM-B) and rJAM-C. S, signal domain; EC, extracellular domain; TM, transmembrane domain; C, cytoplasmic domain. (B) Cell adhesion assay for ADSCs grown on culture dishes coated with the indicated proteins. The relative levels are shown in histograms (mean ± SD; n = 5). (C) BrdU assay for ADSCs grown on culture plates coated with the indicated proteins. The BrdU/DAPI levels are shown in histograms (mean ± SD; n = 12). Scale bar, 100 µm. (D) RT-qPCR for the indicated MSC markers in ADSCs grown on culture dishes coated with the indicated proteins. The relative expression levels are shown in the histograms (mean ± SD; n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Association between soluble JAM-C and full-length JAM-B on ADSCs. Whole-cell lysates (1% of the input protein samples) and the samples immunoprecipitated (IP) with IgG or JAM-B Ab were immunoblotted (IB) with the indicated Abs. N.S., nonspecific signals.

Figure 7

A Soluble JAM-C couples with JAM-B to promote the ADSC adhesion and niche function. (A) Knockdown of the Jam2 gene encoding mouse JAM-B in ADSCs using the CRISPR method. (B) Western blot for the indicated proteins in the whole-cell lysates of the revealed ADSCs. (C) Cell adhesion assay for ADSCs grown in the indicated culture conditions. The relative levels are shown in histograms (mean ± SD; n = 8). (D) RT-qPCR for the indicated MSC markers in ADSCs cultivated in the indicated culture conditions. The relative expression levels are shown in the histograms (mean ± SD; n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 8

Schematic model for the regulation of ADSC functions by soluble JAM-C. The soluble JAM-C (sJAM-C) deposited on the extracellular matrix interacts with JAM-B and partly with JAM-C on ADSC. sJAM-C/JAM-B signaling reaches the nucleus (dashed arrow) and stimulates cellular proliferation and expression of MSC markers (arrows). MSC, mesenchymal stromal/stem cell.