uPA dependent and independent mechanisms of wound healing by C-phycocyanin

Abstract

Wound repair requires both recruitment and well co-ordinated actions of many cell types including inflammatory cells, endothelial cells, epithelial cells and importantly fibroblast cells. Urokinase-type plasminogen activator (uPA) system plays a vital role in wound healing phenomenon. We have previously demonstrated that C-phycocyanin (C-pc), a biliprotein from blue-green algae, transcriptionally regulates uPA through cAMP-dependent protein kinase A (PKA) pathway. To date, a role for C-pc in wound-healing scenario is not elucidated. This study was designed to examine the wound-healing property of C-pc in relation to fibroblast proliferation and migration. C-pc increased fibroblast proliferation in a dose-dependent manner. It also enhanced G1 phase of cell cycle and increased the expressions of cyclin-dependent kinases 1 and 2, which facilitate cell cycle progression, in a uPA-independent manner. In vitro wound healing and migration assays revealed the pro-migratory properties of C-pc. Short-interference RNA studies demonstrated that uPA was necessary for C-pc-induced fibroblast migration. C-pc also significantly elevated the expressions of chemokines (MDC, RANTES, Eotaxin, GRO alpha, ENA78 and TARC) and Rho-GTPases (Cdc 42 and Rac 1) in a uPA-dependent manner. Pre-treatment of C-pc-stimulated cells with pharmacological inhibitor of PI-3K (LY294002) annulled the expression of GTPases implying that Rac 1 and Cdc 42 were induced through PI-3K pathway. C-pc-induced cellular migration towards wounded area was also negatively affected by PI-3K inhibition. In vivo wound-healing experiments in mice validated our finding that C-pc accelerates wound healing. Our data provides conclusive evidence of a novel therapeutic usage for C-pc as a wound-healing agent. C-pc is a food and drug administration (FDA)-approved health supplement. We believe this compound can also be beneficial in healing of internal wounds, such as ulcers.

Fig. 1

Cytotoxic profile of C-pc. Cytotoxicity was measured by quantifying the amount of LDH released from dead cells. C-pc was non-toxic to TIG 3–20 cells upto 100 μg/ml concentration. The effect was similar in normal (uPA⁺)as well as uPA silenced (uPA⁻) cells. Values are mean ±S.D. of three independent experiments.

Fig. 2

Cell viability and proliferation tests. Cell viability is represented by line diagram. Cell proliferation is represented by bar diagram and is denoted by number of cells (× 10⁴ cells/ml). Viability of TIG 3–20 cells was not affected by C-pc up to a dose of 100 μg/ml. C-pc increased cell proliferation in a dose-dependent manner. Values are mean S.D. of three independent experiments.

Fig. 3

Cell cycle analysis. Different phases of cell cycle were analysed by flow cytometry. Treatment with 75 μg/ml C-pc increased the number of cells in the G1 phase. C-pc increased the G1 phase of cell cycle in uPA-independent manner. Values of different cell cycle stage are adjusted to 100%. Results are mean ± S.D. of three independent experiments.

Fig. 4

Expression of cyclin dependent kinases upon stimulation by C-pc: Lysates from uPA⁺ and uPA⁻ cells treated with 75μg/ml C-pc were analyzed by western blot to study the expression of cdK 1 and cdK 2 proteins. Equal loading of proteins was verified using β-actin antibody. Upper panel is a representative image of three independent experiments. The relative density of the proteins was expressed as the ratio of cdK 1/β-actin and cdK 2/β-actin, as shown in lower panel. Values are expressed as mean ± S.D. of three independent experiments. * p < 0.05.

Fig. 5

In vitro wound healing assay: Confluent monolayers of uPA and uPA cells were wounded by gently scratching with a cell scraper and treated with 75 μg/ml C-pc. Photographs were taken 24hrs after wounding. Black line denotes the wound edge at the start of experiments. Wound healing was measured by the distance traveled by the cells from the wound edge. C-pc induced cell migration towards the wounded area in an uPA-dependent manner. *p < 0.05.

Fig. 6

In vitro cell outgrowth assay: Cell outgrowth assay was performed to study the effect of C-pc on random migration of TIG 3–20 cells. uPA⁺ cells stimulated with 75 μg/ml C-pc were significantly more migratory compared to control. The migratory rate was retarded in uPA cells. Values are expressed as mean ± S.D. of three independent experiments. *p < 0.05.

Fig. 7

Effect of C-pc on directed chemotactic migration ofTIG 3–20 cells: Cell migration in response to a range of concentrations of C-pc was measured by Boyden chamber assay. uPA cells responded with higher rate of migration in a dose-dependent manner. C-pc had little effect on uPA⁺ cells. Values are expressed as mean ± S.D. of three independent experiments. *p < 0.05.

Fig. 8

Human chemokine array: After 24 hrs treatment with 75 μg/ml C-pc, supernatants from uPA⁺ (panel A) and uPA⁻ (panel B) cells were analyzed for expression of various chemokines. C-pc induced the expression of a group of chemokines. Values are expressed as mean ± S.D. of three independent experiments. *p < 0.05.

Fig. 9

C-pc induced Cdc 42 and Rac1 in uPA-dependent manner: uPA⁺ and uPA⁻ cells were treated with 75 μg/ml C-pc for 24 hrs and proteins were analyzed by western blotting. -actin was used as control for equal loading. Upper left panel denotes a representative blot of Cdc 42 and upper right panel denotes Rac 1. The relative density of the proteins was expressed as the ratio of Cdc 42/β-actin or Rac 1/β-actin, as shown in lower panel. Data represents the values of three independent values. *p < 0.05

Fig. 10

PI-3K pathway is necessary for C-pc induced expression of Cdc 42 and Rac 1. uPA⁺ cells were pretreated with PI-3K inhibitor, LY294002 (15 nM) for 2 hrs prior to stimulation with 75 μg/ml C-pc. Expression patterns were studied as outlined for Fig. 9. *p < 0.05.

Fig. 11

PI-3K pathway mediates C-pc induced fibroblast migration: uPa⁺ cells were treated with 15 nM LY294002 for 24 h, wounded by gently scratching the monolayer and incubated with 75 μg/ml C-pc for 24 hrs. Black line denotes the wound edge at 0 h. Wound healing was studied as explained in Fig. 5. Data represent mean ± S.D. of three independent experiments. *p < 0.05.

Fig. 12

Dermal wound healing in mice models: C-pc incorporated collagen films were placed over dermal wound as explained in Materials and Methods. Wound healing was measured by quantifying the total wound area over a period of one week. Images shown are representative of three independent experiments. Data represent mean ± S.D.

Fig. 13

Schematic representation of C-pc induced traffic and signal transduction pathway during wound healing.