Basic fibroblast growth factor induces proliferation and collagen production by fibroblasts derived from the bovine corpus luteum†

Abstract

Cyclic regression of the ovarian corpus luteum, the endocrine gland responsible for progesterone production, involves rapid matrix remodeling. Despite fibroblasts in other systems being known for producing and maintaining extracellular matrix, little is known about fibroblasts in the functional or regressing corpus luteum. Vast transcriptomic changes occur in the regressing corpus luteum, among which are reduced levels of vascular endothelial growth factor A (VEGFA) and increased expression of fibroblast growth factor 2 (FGF2) after 4 and 12 h of induced regression, when progesterone is declining and the microvasculature is destabilizing. We hypothesized that FGF2 activates luteal fibroblasts. Analysis of transcriptomic changes during induced luteal regression revealed elevations in markers of fibroblast activation and fibrosis, including fibroblast activation protein (FAP), serpin family E member 1 (SERPINE1), and secreted phosphoprotein 1 (SPP1). To test our hypothesis, we treated bovine luteal fibroblasts with FGF2 to measure downstream signaling, type 1 collagen production, and proliferation. We observed rapid and robust phosphorylation of various signaling pathways involved in proliferation, such as ERK, AKT, and STAT1. From our longer-term treatments, we determined that FGF2 has a concentration-dependent collagen-inducing effect, and that FGF2 acts as a mitogen for luteal fibroblasts. FGF2-induced proliferation was greatly blunted by inhibition of AKT or STAT1 signaling. Our results suggest that luteal fibroblasts are responsive to factors that are released by the regressing bovine corpus luteum, an insight into the contribution of fibroblasts to the microenvironment in the regressing corpus luteum.

Keywords: PGF2α; corpus luteum; fibroblast; fibroblast growth factor; fibrosis; luteolysis.

Graphical Abstract

Figure 1

Fibroblast activation protein (FAP) is increased in regressing corpora lutea. Localization within midcycle and regressed corpora lutea for fibroblast activation protein in corpora lutea obtained from a local abattoir. White arrowheads indicate luteal steroidogenic cells and black arrowheads indicate stained fibroblasts.

Figure 2

Luteal fibroblast growth factors (FGFs) in vivo (A–B) and in vitro (C). Mid-luteal phase cows were injected with saline (Control) or prostaglandin F2α (PGF2α, 25 mg, i.m.) and ovariectomized after 4 and 12 h to collect corpora lutea. RNA sequencing of whole luteal tissue was performed. (A) Most abundant transcripts of FGF family members in the bovine corpus luteum at midcycle and 4- and 12 h post-PGF2α injection. (B) mRNA levels of FGF receptors (FGFRs) at midcycle and 4- and 12 h post-PGF2α injection. Data are presented as mean number of transcripts per million (TPM) ± SEM, n = 4, ^*p < 0.05, ^***p < 0.001, ^****p < 0.0001 compared to 0 h by DESeq2 analysis, Benjamini–Hochberg correction. P values shown are adjusted p values for multiple comparisons. (C) Fibroblasts were treated with or without FGF2 (1 ng/mL) for up to 60 min and cellular lysates prepared for western blot analysis. Shown is a representative experiment repeated in duplicate.

Figure 3

Temporal response to FGF2 on STAT1, ERK, and AKT signaling in bovine luteal fibroblasts. Bovine luteal fibroblasts were treated with 1 ng/mL FGF2 for up to 120 min and protein extracts were processed for western blot analysis of signaling proteins. FGF2 rapidly induces STAT1 (A) and ERK (B) phosphorylation. FGF2 rapidly induces AKT phosphorylation (C) and its downstream targets, GSK3B (D) and p70S6K (E). Statistically significant differences in phosphorylation compared to timepoint 0 were determined by a mixed model analysis with Dunnett’s post hoc multiple comparisons. P values shown are Dunnett’s corrected p values. Data are presented as means ± SEM, n = 5–6 experiments with different fibroblast preparations, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Figure 4

FGF2 increases proliferation of bovine luteal fibroblasts. Cells were treated with varying concentrations of FGF2 for 24 h or 48 h. Proliferation was determined by western blot analysis for the cell proliferation marker PCNA and by direct counting of viable cells by trypan blue staining. (A) Representative PCNA western blot after 24 h of treatment with 0–10 ng/mL of FGF2. (B) Summary of cell counts after 24 h incubation with FGF2 (1 ng/mL). (C) Representative PCNA western blot after 48 h of treatment with FGF2 (0–10 ng/mL). (D) Summary of cell counts after treatment with 0–10 ng/mL of FGF2 for 48 h. Statistically significant differences were identified by mixed model analysis. EdU incorporation assay in the serum-free (E) or 5% serum (F) conditions. The assay was performed as described in the methods. Pink nuclei represent nuclei with EdU incorporation. Cell nuclei were stained with Hoechst (blue). Statistically significant differences between EdU+: total nuclei ratio of control and FGF-treated fibroblasts were calculated by using a paired t-test. Scale bar = 200 μm. Data are represented as means ± SEM, n = 3–5, ^*p < 0.05, ^***p < 0.001, compared to untreated control.

Figure 5

Inhibition of AKT attenuates FGF2-induced proliferation of bovine luteal fibroblasts. Bovine luteal fibroblasts were pretreated for 1 h with vehicle (DMSO) or ERK inhibitor (U0126, 10 μM) and AKT kinase inhibitor (AKTi, 10 μM) prior to treatment with FGF2 (1 ng/mL) for up to 30 min to examine cell signaling events and for up to 72 h for measurements of cell proliferation. (A) Western blot analysis and confirming inhibition of cell signaling. (B) Representative western blot analysis of PCNA and quantification. (C) Cell numbers were determined after 48 h. Data are quantified as a fold change compared to vehicle for its respective timepoint and are presented as mean ± SEM, n = 5, ^*p < 0.05, ^***p < 0.001, ^****p < 0.0001. Statistically significant differences between groups were calculated using a mixed model analysis with Dunnett’s post hoc analysis.

Figure 6

STAT1 activity is required for FGF2-stimulated proliferation of luteal fibroblasts. (A) Bovine luteal fibroblasts were pretreated for 1 h with the MTOR inhibitor rapamycin (Rap, 50 nM) or the STAT1 inhibitor fludarabine (Flu, 100 μM) and then incubated without or with FGF2 (1 ng/mL) for 24–72 h (B). The proliferation marker PCNA was measured by western blot analysis. β-Actin (ACTB) was used as a loading control, n = 4. (C) Proliferation was also measured by cell counting via trypan blue exclusion after treatment with FGF2 for 48 h. Data are quantified as a fold change compared to vehicle for its respective timepoint and are presented as mean ± SEM, n = 4. Dunnett’s corrected p values were determined by mixed model analysis, ^*p < 0.05, ^***p < 0.001, ^****p < 0.0001.

Figure 7

FGF2 increases collagen production by bovine luteal fibroblasts. Bovine luteal fibroblasts were treated without or with increasing concentrations of FGF2 (0–10 ng/mL) for 48 h. Treatment with TGFB1 (1 ng/ml) was used as a positive control. (A) Representative western blot of type 1 collagen. β-Actin (ACTB) was used as a loading control. (B) Quantification of collagen production. Data are quantified as a fold change compared to control (no FGF2) and presented as mean ± SEM, n = 3. Statistical differences between groups were calculated by a mixed model analysis with Dunnett’s post hoc analysis. Dunnett’s corrected p values ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.