Cell proliferation in in vivo-like three-dimensional cell culture is regulated by sequestration of ERK1/2 to lipid rafts

Abstract

Objectives: Regulatory mechanisms of cell proliferation have been extensively studied as they represent major challenges when dealing with pathologies such as fibrosis, tumourigenesis or tissue regeneration. Numerous in vitro studies still exploit conventional, two-dimensional cell cultures where cells are forced to adhere to unnaturally stiff and flat surfaces of culture dishes. In the living organism, however, each cell is in contact with components of the extracellular matrix and/or neighbouring cells, thus creating a complex three-dimensional (3D) tissue structure. The current paper describes a native 3D culture of cells, based on the GD25β1 fibroblast cell line, and its use for investigating cell proliferation in in vivo-like conditions.

Materials and methods: Four-day post-confluent culture of GD25β1 fibroblasts resulted in formation of a 3D system of cells embedded in naturally synthesized extracellular matrix. Morphological characterization of the culture was performed by histochemistry, immunohistochemistry and immunofluorescence. Viability/proliferation was assayed by MTT testing, FACS analysis and Western blotting for determination of expression levels and activation status of the relevant signalling molecules.

Results: GD25b1 fibroblasts, grown as 3D culture, gave rise to tissue-like structures characterized by low level of apoptosis, low senescence and development of 3D matrix adhesions, typical of living tissues. Transition to three-dimensionality led to a switch from exponential to linear culture growth, accompanied by accumulation of activated ERK1/2 into caveolin-containing raft domains. Disruption of raft domains as well as reverse transition from 3D back to monolayer culture led to release of phosphorylated ERK1/2 from rafts, activation of cyclin D1 expression and increase in proliferation levels.

Conclusions: These results imply that under in vivo-like conditions, cells might achieve reduction of their proliferation level by sequestering activated ERK1/2 to lipid rafts.

Figure 1

Characterization of the three‐dimensional in vivo ‐like culture, formed by GD25β1 fibroblasts. (a) Growth curve of GD25β1 fibroblasts as 3D culture, determined by MTT assay. Dotted line represents calculated linear growth curve. (b) Haematoxylin/eosin staining of paraffin wax cross‐sections through 3D culture (upper panel) and Coomasie Brilliant Blue staining of decellularized 3D culture (lower panel). (c) Immunofluorescent staining of monolayer (2D) and three‐dimensional (3D) culture with antibody against activated β1 integrin (anti‐β1 integrin). Insets demonstrate general morphology of GD25β1 fibroblasts under 2D and 3D conditions. (d) β ‐galactosidase staining of GD25β1 fibroblasts from monolayer (2D) and three‐dimensional (3D) culture. P‐cell line past 25th passage (P cells) used as a positive control. Blue stained cells represent positive for beta‐galactosidase cells. Graph representing percentage of positive cells in 2D and 3D cultures. Error bars represent ±SE. Results are from at least three independent experiments.

Figure 2

Proliferation features of the three‐dimensional culture. (a) FACS analysis of CSFE stained GD25β1 fibroblasts before plating (stained cells) and after formation of 3D culture (stained 3D cells). (b) Immunohistochemical staining with anti‐cyclin D antibodies of paraffin wax embedded sections from control monolayer culture (2D) and three‐dimensional culture (3D) of GD25β1 fibroblasts. Sections were counterstained with methyl green for visualization of nuclei. Cyclin D positive nuclei appear dark brown (arrows). (c) Western blot analysis of level of activated ERK1/2 (pERK1/2) and expression of cyclin D (cyclin D) in samples prepared at different time points (days in culture) of development of the 3D culture. Internal controls, performed with antibodies against total ERK1/2 (tERK1/2) and tubulin (Tubulin), were used for normalization of loading and determination of fold change of studied molecules (graph). Error bars represent ±SE. Results from at least three independent experiments.

Figure 3

Relationship between proliferation in 3D cell culture and the amount of phosphorylated ERK1/2 localized at lipid rafts. Western blot determination and quantification of the amount of: (a) Total ERK1/2 (tERK1/2) in detergent‐insoluble fraction (Rafts) isolated from monolayer (2D) and three‐dimensional (3D) culture of GD25β1 fibroblasts. Flotillin was used as internal control for determination of the fold change of the amount of total ERK1/2 (graph); (b) Phosphorylated ERK1/2 (pERK1/2) in detergent‐insoluble fraction (Rafts) isolated from monolayer (2D) and three‐dimensional (3D) culture of GD25β1 fibroblasts. Flotillin was used as internal control for determination of the fold change of the amount of phosphorylated ERK1/2 (graph); (c) Phosphorylated ERK1/2 (pERK1/2) in detergent‐insoluble fraction (Rafts) isolated from control three‐dimensional culture (3D) or 3D culture treated with lovastatin (3d + LS). Flotillin was used as internal control for determination of the fold change of the amount of phosphorylated ERK1/2 (graph); (d) Cyclin D in control three‐dimensional culture (3D) or 3D culture treated with lovastatin (3d + LS). Tubulin was used as internal control for determination of the fold change of cyclin D expression (graph); (e) Phosphorylated ERK1/2 (pERK1/2) in detergent‐insoluble fraction (Rafts) isolated from three‐dimensional (3D) culture or cells from 3D culture plated as monolayer for the indicated time periods (days in 2D). Flotillin was used as internal control for determination of the fold change of the amount of phosphorylated ERK1/2 (graph). (f) Growth curve of monolayer GD25β1 fibroblasts (2D cells) and GD25β1 fibroblasts from three‐dimensional culture (3D cells) plated under conventional monolayer conditions. Error bars represent ±SE. The results are from at least three independent experiments.