Functional melanocortin-2 receptors are expressed by mouse aorta-derived mesenchymal progenitor cells

Abstract

A local melanocortin system is active during tissue injury and inflammation. Thus far this system has been described as autocrine in nature where local production of pro-opiomelanocortin (POMC) peptides by leukocytes feeds back on melanocortin receptor (MC-R) expressing immune cells to quell inflammatory cytokine production. Here we present evidence that POMC peptides may generate extracellular matrix (ECM) changes by inducing matrix production by cells of the mesenchymal lineage through activation of the MC2-R. Using immunoblot, we determined that mouse aorta-derived mesenchymal progenitor cells express both MC2-R and MC3-R. These progenitors respond to treatment with ACTH by increasing collagen matrix synthesis as assessed by picrosirius red stain and (3)H-proline incorporation. ACTH also induces transient increases in intracellular calcium ([Ca(2+)](i)) as assessed using the fluorescent Ca(2+) indicator, fura-2. The ACTH-induced changes in [Ca(2+)](i) are consistent with MC2-R signaling and consist of both an intracellular release and an extracellular influx of Ca(2+). Both mouse aortic mesenchymal progenitors and mouse macrophage cells express POMC and the prohormone convertase 1/3 (PC1/3) indicating they have the potential to contribute to the local production of POMC peptides. These data demonstrate functional MC2-R expression in mouse aorta-derived mesenchymal progenitors and implicate both macrophage and mesenchymal cells as relevant sources of local POMC peptides.

Fig. 1

Immunofluorescent detection of mesenchymal cell markers in aorta-derived cell lines. The clonal cell line (C8) and the late passage (mAo) cell lines are positive for fibronectin, integrin β1 and vimentin. Digital multi-channel fluorescent images were taken at a 400× magnification using a Nikon A1 confocal microscope, bar = 10 µM.

Fig. 2

Adipogenic and osteogenic lineage differentiation of aortic mesenchymal progenitor cell lines. The mAo late passage cell line was capable of adipogenic differentiation with induction (A) as assessed by oil-red-O stain. Both the mAo (B) and the C8 clonal cell line (C) were capable of mineralization as assessed by von Kossa stain. Bars = 100 µM

Fig. 3

ACTH reduces proliferation and increases proline incorporation in aorta-derived mesenchymal progenitor cell lines. Proliferation was assessed using MTT assay in mAo (A) and C8 cultures (B). Cells were plated at 7500 cells per well in 96-well plates. After 24 h (day 0), MTT was added and after 3 h and precipitated formazan was solubilized and absorbance read at 550 nm. On day 0, additional 96-well plates were treated with and without ACTH (1–39) at 10 nM, 100 nM and 1 µM. Medium was changed every 2–3 days and during the last 3 h of incubation on days 1, 4 and 7, MTT was added, incubated for 3 h and precipitated formazan was solubilized and absorbance read at 550 nm. Data were expressed as fold change in OD from day 0. Results are presented as mean ± SD, n=6–12.

Fig. 4

Collagen synthesis is increased in mAo MSC after exposure to ACTH. Collagen production was examined by picrosirius red stain (A & B) and by ³H-proline incorporation (C). For quantitation of picrosirius red stain (B) the threshold intensity used was ≥80% the highest intensity in the untreated control (0) and 4 fields per well analyzed, n = 4. For ³H-proline incorporation (C), data are presented as mean ± SD, n = 3 wells. Statistically significant differences between individual treatments were determined after a significant one-way ANOVA using the Bonferonni correction with a P < 0.05 considered significant. *- significantly different from 0. Data are representative of 3 separate experiments with similar results.

Fig. 5

Immunoblot results showing MC2-R and MC3-R expression in aorta-derived mesenchymal cell lines. (A) A representative blot showing MC2-R detection in crude membrane fractions of mouse aorta cells mAo) untreated (−) or treated (+) with 10 nM Dex; the clonal mouse aorta-derived cell line (C8) untreated (−) or treated (+) with 10 nM Dex; and rat bone marrow derived MSC induced to form adipocytes (Adip) as a control (Adip con). (B) A representative blot showing detection of MC3-R in crude membrane fractions of mAo cells untreated (−) or treated (+) with 10 nM Dex and C8 cells untreated (−) or treated (+) with 10 nM Dex. Adip were used as a negative control (neg con). (C) A representative blot showing the absence of MC5-R in crude membrane fractions of mAo cells untreated (lane 1) or treated with 10 nM Dex (lane 2) and C8 cells untreated untreated (−) or treated (+) with 10 nM Dex Adip were used as a positive control (pos con). SDS-PAGE gels were loaded with 50 µg protein crude membrane fraction per lane. Detection of Na⁺/K⁺ ATPase was used as a control for gel loading. (D) Densitometric analysis of MC2-R and MC3-R immunoblots. Data are presented as mean % expression ± SD of untreated (− Dex) cultures, n = 3–4.

Fig. 6

The clonal aorta-derived mesenchymal progenitor cell line, C8, responds to ACTH with transient increases in intracellular calcium ([Ca²⁺]_i) and these increases are significantly enhanced with dexamethasone priming. (A) Representative fluorimetric traces of calcium flux are shown in cultures left untreated (−Dex) or treated with 10 nM dexamethasone (+Dex) for 24 h. The arrow shows the time of ACTH addition and dose is presented under the corresponding trace. (B) The quantitative comparison of calcium flux shown in A. Clear bars: −DEX, Solid bars: +DEX. Statistically significant differences between individual treatments were determined after a significant two-way ANOVA using the Bonferonni correction with a P < 0.05 considered significant, n = 3–4, a- significantly different from −DEX counterpart, b- significantly different from 500 nM ACTH counterpart.

Fig. 7

Mouse aorta-derived mesenchymal progenitor cells, mAo cells, respond to ACTH with transient increases in intracellular calcium ([Ca²⁺]_i) in a dose-dependent manner (A & B) and these increases have both an intracellular and extracellular contribution (C & D). In (A) representative fluorimetric traces of the dose-dependent, ACTH-induced transient elevations in [Ca²⁺]_i in the presence of extracellular calcium are shown. The arrow shows the time of ACTH addition and the dose is presented under or to the right of the corresponding trace. In (B) the quantitative comparison of calcium flux represented in A is shown. Data are presented as the mean change in [Ca²⁺]_i from baseline ± SD, n = 4–5. In (C) representative traces of ACTH-induced transient elevations in [Ca²⁺]_i in the absence of extracellular calcium are shown along with the influx of calcium after the re-addition of 1.8 mM CaCl₂ to the medium. The arrows show the times of ACTH and CaCl₂ addition. The initial dose of ACTH ([ACTH]init) is shown to the right of the corresponding trace. In (D) the quantitative comparison of calcium flux represented in C is shown. Clear bars represent mean ± SD of intracellular calcium release (IC) and the solid bars represent the mean ± SD of the extracellular influx (EC), n = 4. a -significantly different from 10 and 0 nM counterpart, b -significantly different from 100, 10 and 0 nM counterpart.

Fig. 8

MC3-R agonists do not induce calcium flux in aorta-derived mesenchymal cell lines. The C8 and mAo cell lines were loaded with the fura-2 Ca²⁺ indicator and responses to γ₂-MSH (1 µM) and MTII (1 µM) were measured in the presence of extracellular calcium. The arrows show the time of peptide addition.

Fig. 9

PC1/3 and POMC expression in aorta-derived mesenchymal cell lines and activation of murine macrophage regulates PC1/3 and POMC expression. Twenty-five micrograms of total protein lysate from mAo, C8 and mouse macrophage (mMa) cells were analyzed for POMC and PC1/3 expression via immunoblot.