Salamander-derived, human-optimized nAG protein suppresses collagen synthesis and increases collagen degradation in primary human fibroblasts

Abstract

Unlike humans, salamanders regrow their amputated limbs. Regeneration depends on the presence of regenerating axons which upregulate the expression of newt anterior gradient (nAG) protein. We had the hypothesis that nAG might have an inhibitory effect on collagen production since excessive collagen production results in scarring, which is a major enemy to regeneration. nAG gene was designed, synthesized, and cloned. The cloned vector was then transfected into primary human fibroblasts. The results showed that the expression of nAG protein in primary human fibroblast cells suppresses the expression of collagen I and III, with or without TGF- β 1 stimulation. This suppression is due to a dual effect of nAG both by decreasing collagen synthesis and by increasing collagen degradation. Furthermore, nAG had an inhibitory effect on proliferation of transfected fibroblasts. It was concluded that nAG suppresses collagen through multiple effects.

Figure 1

Identification of nAG gene in mammalian expression plasmid nAG-pJexpress pJ608. (a) The plasmid contains nAG gene, optimized for human cell expression and tagged with V5 peptide for further identification. The gene is surrounded by two restriction sites: XhoI and Not I. (b) nAG plasmid integrity was confirmed by DNA electrophoresis showing the plasmid in two forms: circular uncut plasmid and linearized cut with XhoI.

Figure 2

Western blot assay (a) and immunofluorescence (b) experiments showing nAG protein expression in primary human fibroblasts. (a) Western blot was performed after 24 h and 48 h of transfection. The cells were lysed by using RIPA cocktail; proteins were separated on 12% SDS polyacrylamide gel. The primary antibody V5 probe and HRP-conjugated secondary antibody were used for nAG protein detection in the following lanes: nontransfected fibroblasts (negative control 1), fibroblasts with nAG plasmid without lipofection (negative control 2), nAG transfected fibroblasts tested after 24 h and nAG transfected fibroblasts tested after 48 h. (b) Immunofluorescence assay was performed after 48 h of transfection and cells were fixed and permeabilized by 2% PFA/0.1% Triton x-100. V5 probe was used as primary antibody and green fluorescence was detected by using FITC-conjugated secondary antibody. Compare nAG protein expression in nontransfected fibroblasts (negative control) to nAG expression in transfected fibroblasts (magnification 40x).

Figure 3

BrdU incorporation ELISA assay for assessment of proliferation activity in nontransfected and nAG transfected fibroblasts. The cells were cultured in 96-well plates at a density of 8000 cells/well. 24 and 48 hours after lipofection, cells were assayed for proliferation by measuring BrdU incorporation during DNA synthesis in proliferating cells. The results showed the inhibitory effect of nAG on fibroblasts proliferation, after 24 h by 47% decrease (P < 0.0001) and after 48 h by 42% decrease (P < 0.0001) in proliferation in nAG transfected fibroblasts compared to nontransfected fibroblasts.

Figure 4

Western blot experiment for detection of collagen I (a) and collagen III (b) expression in primary human fibroblasts transfected with nAG plasmid with or without treatment with TGF-β1. Cells were lysed after 48 hours using RIPA cocktail. Proteins were separated on 7.5% SDS polyacrylamide gel and detected by using COL1A1 (primary antibody for collagen I), COL3A1 (primary antibody for collagen III), and HRP-conjugated secondary antibody and the immunoblots were visualized by using ECL kit. (a) Collagen I detection: lane-1: collagen I expression in non-transfected fibroblasts without any treatment (control 1), lane-2: collagen I expression in fibroblasts transfected with nAG plasmid, lane-3: collagen I expression in non-transfected fibroblasts treated with TGF-β1 (control 2), lane-4: collagen I expression in fibroblasts transfected with nAG plasmid and treated with TGF-β1, and lanes-5–8: internal control β-actin. (b) Collagen III detection: lane-1: collagen III expression in non-transfected fibroblasts without any treatment (control 1), lane-2: collagen III expression in non-transfected fibroblasts with TGF-β1 treatment (control 2), lane-3: collagen III expression in fibroblasts transfected with nAG plasmid, lane-4: collagen III expression in fibroblasts transfected with nAG plasmid and treated with TGF-β1, and lanes-5 to 8 are internal control β-actin.

Figure 5

Immunofluorescence experiment showing effect of nAG on collagen I (a) and collagen III (b) expression in primary human fibroblasts with or without TGF-β1 treatment. After 48 h of transfection, immunoflourescence was performed by using COL1A1, COL3A1 (primary antibodies), and FITC-conjugated secondary antibody. (a) Immunofluorescence staining of type I collagen is in the following order: control fibroblasts (complete medium + 150 μg/mL L-ascorbic acid), control fibroblasts treated with 3 ng/mL TGF-β1, fibroblasts with nAG plasmid, and fibroblasts with nAG plasmid and treated with 3 ng/mL TGF-β1 (magnification 40x). Note the suppressive effect of nAG on collagen I. (b) Immunofluorescence staining of type III collagen is in the following order: control fibroblasts (complete medium + 150 μg/mL L-ascorbic acid), control fibroblasts treated with 10 ng/mL TGF-β1, fibroblasts with nAG plasmid, and fibroblasts with nAG plasmid and treated with 10 ng/mL TGF-β1 (magnification 40x). Note the complete suppressive effect of nAG on collagen III.

Figure 6

Quantitative real-time PCR (RT-PCR) measuring relative mRNA expressions level of procollagen I and procollagen III in non-transfected and nAG transfected primary human fibroblasts. 100 ng of total RNA was reverse-transcribed and target genes expression was measured in multiplex, one-step RT-PCR by using TaqMan probes with (FAM, HEX, or ROX) reporter dyes and (BHQ1 or BHQ2) quencher. To estimate effect of nAG on collagen I and collagen III synthesis, the relative mRNA expressions were related to the reference gene, β-actin. The relative expression of procollagen I was 55% decrease (P < 0.001) and procollagen III was 95% decrease (P < 0.0001) in nAG transfected fibroblasts compared to non-transfected fibroblasts. The data represents the mean of three independent experiments.

Figure 7

Determination of pro-MMP-2 and active MMP-2 by gelatin zymography ((a) and (b)) and pro-MMP-1 by ELISA (c) in nontransfected and nAG transfected fibroblasts. (a) Gelatin zymography: media were harvested after two days of transfection of nAG plasmid into fibroblasts and non-transfected fibroblasts (both were treated with 3 ng/mL TGF-β1). The first three lanes were different fractions of the same sample of non-transfected fibroblasts, the fourth lane was MMP-2 (positive control), and the last three lanes were samples of nAG transfected fibroblasts. (b) Densitometry analysis for gelatin zymography: density of each band was measured by using densitometry tool in Gel documentation software. There was 37% increase in pro-MMP-2 (P < 0.019) and 85% increase in active MMP-2 (P < 0.001) in transfected fibroblasts compared to non-transfected fibroblasts. (c) ELISA assay for pro-MMP-1 measurement: after two days of transfection. Media were harvested for measurement of pro-MMP-1 in non-transfected fibroblasts (control), nAG transfected fibroblasts, and fibroblasts treated with recombinant nAG. The level of pro-MMP-1 was 53-fold increase in transfected fibroblast compared to non-transfected fibroblasts (P < 0.004) and it was 4-fold increase in nAG-treated fibroblasts compared to non-transfected fibroblasts (P < 0.0001).