Human uterine leiomyoma-derived fibroblasts stimulate uterine leiomyoma cell proliferation and collagen type I production, and activate RTKs and TGF beta receptor signaling in coculture

Alicia B Moore, Linda Yu, Carol D Swartz, Xaiolin Zheng, Lu Wang, Lysandra Castro, Grace E Kissling, David K Walmer, Stanley J Robboy, Darlene Dixon, Alicia B Moore, Linda Yu, Carol D Swartz, Xaiolin Zheng, Lu Wang, Lysandra Castro, Grace E Kissling, David K Walmer, Stanley J Robboy, Darlene Dixon

Abstract

Background: Uterine leiomyomas (fibroids) are benign smooth muscle tumors that often contain an excessive extracellular matrix (ECM). In the present study, we investigated the interactions between human uterine leiomyoma (UtLM) cells and uterine leiomyoma-derived fibroblasts (FB), and their importance in cell growth and ECM protein production using a coculture system.

Results: We found enhanced cell proliferation, and elevated levels of ECM collagen type I and insulin-like growth factor-binding protein-3 after coculturing. There was also increased secretion of vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor-2, and platelet derived growth factor A and B in the media of UtLM cells cocultured with FB. Protein arrays revealed increased phosphorylated receptor tyrosine kinases (RTKs) of the above growth factor ligands, and immunoblots showed elevated levels of the RTK downstream effector, phospho-mitogen activated protein kinase 44/42 in cocultured UtLM cells. There was also increased secretion of transforming growth factor-beta 1 and 3, and immunoprecipitated transforming growth factor-beta receptor I from cocultured UtLM cells showed elevated phosphoserine expression. The downstream effectors phospho-small mothers against decapentaplegic -2 and -3 protein (SMAD) levels were also increased in cocultured UtLM cells. However, none of the above effects were seen in normal myometrial cells cocultured with FB. The soluble factors released by tumor-derived fibroblasts and/or UtLM cells, and activation of the growth factor receptors and their pathways stimulated the proliferation of UtLM cells and enhanced the production of ECM proteins.

Conclusions: These data support the importance of interactions between fibroid tumor cells and ECM fibroblasts in vivo, and the role of growth factors, and ECM proteins in the pathogenesis of uterine fibroids.

Figures

Figure 1
Figure 1
Desmin and Vimentin. (A) Uterine leiomyoma-derived fibroblasts (FB) stained negatively for desmin (center) and positively for vimentin (upper left). Inset lower right: Negative Control (NC) = Normal Mouse Serum. (B) UtLM cells and UtSMC (C) stained positively for desmin (center) and vimentin (upper left). Inset lower right: NC = Normal Mouse Serum. OTR. (D) Leiomyoma-derived FB was negative for the OTR. Inset upper left: NC = Normal Goat Serum. Positive staining of the OTR was observed in both UtLM cells (E) and UtSMC (F). Inset upper left: NC = Normal Goat Serum.
Figure 2
Figure 2
Assessment of cell proliferation based on cell counts and PCNA labeling. (A) Cell proliferation. There was a significant (p = 0.004) increase in the number of UtLM cells cocultured with fibroblasts (UtLM/FB) compared to UtLM cells cultured alone. There was no significant difference observed between UtSMC alone and UtSMC cocultured with FB (B) PCNA labeling. The percent PCNA labeling was significantly (p = 0.04) higher in UtLM/FB compared to UtLM cells cultured alone; however, there was no significant difference for UtSMC at both conditions. (C) PCNA immunoexpression in UtLM cells and UtLM/FB. PCNA was expressed in both cocultured and non-cocultured UtLM cells, as well as UtSMC, as indicated by brown nuclear staining. There was a significant increase in PCNA expression in the UtLM/FB compared to UtLM cells cultured alone, but not for UtSMC under similar conditions. All error bars represent SEM.
Figure 3
Figure 3
ECM Proteins. (A) Western blots. Collagen-I and IGF-BP-3 protein secretion in media of UtLM cells or UtSCM cultured in the presence (UtLM/FB or UtSMC/FB) or absence of FB. (B) Densitometry. Collagen I was significantly (p = 0.02) increased in the media of UtLM/FB cells compared to UtLM cells cultured alone. There was no difference between UtSMC versus UtSMC cocultured with FB. There was a significant (p = 0.02) increase in the expression of IGF-BP-3 in the cocultured UtLM cells compared to UtLM cells or FB cultured alone. Furthermore, IGF-BP-3 levels were also significantly higher in the cocultured UtSMC than UtSMC and FB cultured alone, but the level was minimal. There was a limited expression of IGF-BP-4 compared to IGF-BP-3 for both UtLM cells and UtSMC under cocultured or single cultured conditions. However, there was a significant (p = 0.02) increase in IGF-BP-4 expression in the cocultured UtSMC compared to UtSMC cultured alone. All error bars represent SEM.
Figure 4
Figure 4
Secretion of TGF-β1, VEGF and EGF in the media. There was a significant increase in the concentration of (A) TGF-β1 (p = 0.01), (B) VEGF (p = 0.02) and (C) EGF (p = 0.004) in the media of UtLM/FB compared to UtLM cells or fibroblasts (FB) cultured alone by ELISA. All error bars represent SEM.
Figure 5
Figure 5
Secretion of FGF-2, PDGF-A and -B, and TGF-β3 in the media. (A) Western blots of growth factor proteins. (B) Densitometric intensity of growth factor proteins. There was a statistically significant (p = 0.02) increase in the secretion of FGF-2, PDGF-A and -B and TGF-β3 in the media of UtLM cells cocultured with fibroblasts (UtLM/FB) versus UtLM cells or FB cultured alone. All error bars represent SEM.
Figure 6
Figure 6
Expression of phosphorylated (p)RTKs. (A) pRTK array. The array detected activated RTKs for GFs under both culturing conditions (B) Densitometry. There was an overall increase in the expression of 5 pRTKs in the UtLM/FB compared to UtLM cells cultured alone. Significant (p = 0.02) increases in the phosphorylated receptors, ErbB4 and PDGFα/β, were found in the UtLM/FB compared to UtLM cells cultured alone. All error bars represent SEM.
Figure 7
Figure 7
Detection of phosphorylated (p)-MAPK44/42. (A) Western blots. There was a significant increase in the expression of p-MAPK44/42 in the UtLM/FB compared to cultures of UtLM cells or FB alone at 24 h, 48h and day 7. (B) Densitometry. There was a significant increase in the expression of p-MAPK44/42 in cocultured UtLM cells compared to non-cocultured UtLM cells and FB at 24 h (p < 0.0001), 48 h (p = 0.05) and day 7 (p = 0.05). There were no differences observed between the culturing conditions for total (t)-MAPK44/42 expression. All error bars represent SEM.
Figure 8
Figure 8
Detection of activated TGF-βRI and downstream proteins, p-Smad-2 and -3 at 48 h. (A) Immunoprecipitation of TGF-βRI, blotted with anti-phospho-serine. There was increased expression of phospho-serine in immunoprecipitated TGF-βRI in cocultures of UtLM/FB compared to cultures of FB or UtLM cells alone. There were no significant differences in total (t)-TGF-βRI expression between the culturing conditions. (b) Western blots of p-Smad-2 and p-Smad-3 and t-Smad-2 and t-Smad-3. Densitometric analyses showed a significant increase in the expression of phospho-Smad-2 (p = 0.001) and phosho-Smad-3 (p = 0.05) in UtLM/FB compared to UtLM cells or FB cultured alone. There was no significant difference in t-Smad-2 expression between UtLM/FB and UtLM cells cultured alone. All error bars represent SEM.

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