Mechanical homeostasis is altered in uterine leiomyoma

Abstract

Objective: Uterine leiomyoma produce an extracellular matrix (ECM) that is abnormal in its volume, content, and structure. Alterations in ECM can modify mechanical stress on cells and lead to activation of Rho-dependent signaling and cell growth. Here we sought to determine whether the altered ECM that is produced by leiomyoma was accompanied by an altered state of mechanical homeostasis.

Study design: We measured the mechanical response of paired leiomyoma and myometrial samples and performed immunogold, confocal microscopy, and immunohistochemical analyses.

Results: Leiomyoma were significantly stiffer than matched myometrium. The increased stiffness was accompanied by alteration of the ECM, cell shape, and cytoskeleton in leiomyoma, compared with myometrial samples from the same uterus. Levels of AKAP13, a protein that is known to activate Rho, were increased in leiomyoma compared to myometrium. AKAP13 was associated with cytoskeletal filaments of immortalized leiomyoma cells.

Conclusion: Leiomyoma cells are exposed to increased mechanical loading and show structural and biochemical features that are consistent with the activation of solid-state signaling.

Figure 1. Mechanical testing in matched surgical specimens of uterine leiomyoma and myometrium

1A: Measurement of compressive resistance to 10% strain (Young’s modulus) in leiomyoma and myometrial samples. Y=kilopascals (kPa), mean±SEM. X=samples of myometrium (M) or leiomyoma (L). Results were replicated in four independent studies. 1B: Measurement of total sulfated GAG content in matched leiomyoma and myometrial samples analyzed by compression using the blyscan method for wet (left panel) and dry weight (right panel). Y axes= micrograms of GAG per microgram of sample. X axes=sample myometrium (M) or leiomyoma (L). Results shown are averages of 3 representative assays. 1D: Hydroxyproline measurement in matched leiomyoma (L) and myometrial (M) samples corrected for wet and dry weight, as shown. Results shown are representative of 3 assays.

Figure 1. Mechanical testing in matched surgical specimens of uterine leiomyoma and myometrium

1A: Measurement of compressive resistance to 10% strain (Young’s modulus) in leiomyoma and myometrial samples. Y=kilopascals (kPa), mean±SEM. X=samples of myometrium (M) or leiomyoma (L). Results were replicated in four independent studies. 1B: Measurement of total sulfated GAG content in matched leiomyoma and myometrial samples analyzed by compression using the blyscan method for wet (left panel) and dry weight (right panel). Y axes= micrograms of GAG per microgram of sample. X axes=sample myometrium (M) or leiomyoma (L). Results shown are averages of 3 representative assays. 1D: Hydroxyproline measurement in matched leiomyoma (L) and myometrial (M) samples corrected for wet and dry weight, as shown. Results shown are representative of 3 assays.

Figure 2. Structural changes associated with uterine leiomyoma

2A–D: FITC-Phalloidin staining of sections from matched leiomyoma and myometrial samples. Staining for actin in fibroid samples reveals a disordered structure and is markedly different from normal myometrium. Leiomyoma (A) and myometrium (B) were from the same uterus; leiomyoma (C) and myometrium (D) were from a separate specimen. Note the deformed nuclei and cell structure in A and C. Representative studies. Magnification 40X. 2 E,F: Staining of paired leiomyoma (E) and myometrium (F) for RhoA (1:1000) suggested increased staining. Magnification=40X. 2 G,H: Staining of paired leiomyoma (G) and myometrial (H) sections for alpha-smooth muscle actin (1:1500). Expression was slightly increased in leiomyoma cells. Magnification=40x

Figure 2. Structural changes associated with uterine leiomyoma

2A–D: FITC-Phalloidin staining of sections from matched leiomyoma and myometrial samples. Staining for actin in fibroid samples reveals a disordered structure and is markedly different from normal myometrium. Leiomyoma (A) and myometrium (B) were from the same uterus; leiomyoma (C) and myometrium (D) were from a separate specimen. Note the deformed nuclei and cell structure in A and C. Representative studies. Magnification 40X. 2 E,F: Staining of paired leiomyoma (E) and myometrium (F) for RhoA (1:1000) suggested increased staining. Magnification=40X. 2 G,H: Staining of paired leiomyoma (G) and myometrial (H) sections for alpha-smooth muscle actin (1:1500). Expression was slightly increased in leiomyoma cells. Magnification=40x

Figure 3. Altered expression of factors involved in mechanical transduction in matched surgical specimens of uterine leiomyoma and myometrium

3A: Western analysis of leiomyoma (L) and myometrial (M) lysates for expression of the Rho-GEF AKAP13 using 2665 affinity-purified antisera. A 220kDa band was present that was increased in leiomyoma compared to myometrium. Samples from patients 12–16, 40–41, 50, 51 were used for all western blots. 3B: Beta actin control for lysates in A. 3C: Western analysis for AKAP13 using monoclonal antibody directed against Brx (Upstate, Temecula, CA). A 220kDa band is identified. Positive control (+) consisted of lysates prepared from Cos-7 cells transfected with a construct expressing a 170kDa form of AKAP13. The negative control (−), lane 12, were lysates prepared from un-transfected Cos-7 cells (Cos-7 cells do not express AKAP13). 3D: Western analysis for AKAP13 in matched leiomyoma (L) and myometrial (M) lysates from 8 patients. In most, but not all pairs, expression of AKAP13 was greater in leiomyoma (L) compared to myometrium (M). 3E: Western analysis for RhoA in the same lysates. Readily extractable levels of RhoA were increased in some, but not all fibroid samples. 3F: Beta actin control for protein loading.

Figure 3. Altered expression of factors involved in mechanical transduction in matched surgical specimens of uterine leiomyoma and myometrium

3A: Western analysis of leiomyoma (L) and myometrial (M) lysates for expression of the Rho-GEF AKAP13 using 2665 affinity-purified antisera. A 220kDa band was present that was increased in leiomyoma compared to myometrium. Samples from patients 12–16, 40–41, 50, 51 were used for all western blots. 3B: Beta actin control for lysates in A. 3C: Western analysis for AKAP13 using monoclonal antibody directed against Brx (Upstate, Temecula, CA). A 220kDa band is identified. Positive control (+) consisted of lysates prepared from Cos-7 cells transfected with a construct expressing a 170kDa form of AKAP13. The negative control (−), lane 12, were lysates prepared from un-transfected Cos-7 cells (Cos-7 cells do not express AKAP13). 3D: Western analysis for AKAP13 in matched leiomyoma (L) and myometrial (M) lysates from 8 patients. In most, but not all pairs, expression of AKAP13 was greater in leiomyoma (L) compared to myometrium (M). 3E: Western analysis for RhoA in the same lysates. Readily extractable levels of RhoA were increased in some, but not all fibroid samples. 3F: Beta actin control for protein loading.

Figure 4. Subcellular distribution of AKAP13 in matched leiomyoma and myometrial specimens

4 A–D: Immunohistochemical localization of AKAP1 (1:500) in leiomyoma (A) and myometrium (B) tissues. Magnification=63X. Positive control, breast tissue (C); negative control=fibroid tissue stained with pre-immune antisera (D). Staining for AKAP13 was often appeared increased with a peri-nuclear appearance in fibroids. 4 E, F: Immunogold study of matched leiomyoma (E) and myometrium (F) specimens using 2665 anti-sera directed against AKAP13. Note the angular cell shape, reduced cytoplasm, and notched nucleus in the leiomyoma (E) compared to myometrium (F). Round black dots indicate localization of protein. Black triangle points to nucleus. Cyt=cytoplasm; ECM=extracellular matrix. Magnification=21,000X. Samples from patients 18–21 were used for immunogold experiments. 4 G, H: Immunogold staining from another matched pair of leiomyoma (G) and myometrial (H) tissues stained with antisera directed against AKAP13. In this view, arrows point to AKAP13 expression in the nucleus, nuclear envelope and cytoplasm. Magnification=15,500X. Results were repeated in matched samples from 4 patients.

Figure 4. Subcellular distribution of AKAP13 in matched leiomyoma and myometrial specimens

4 A–D: Immunohistochemical localization of AKAP1 (1:500) in leiomyoma (A) and myometrium (B) tissues. Magnification=63X. Positive control, breast tissue (C); negative control=fibroid tissue stained with pre-immune antisera (D). Staining for AKAP13 was often appeared increased with a peri-nuclear appearance in fibroids. 4 E, F: Immunogold study of matched leiomyoma (E) and myometrium (F) specimens using 2665 anti-sera directed against AKAP13. Note the angular cell shape, reduced cytoplasm, and notched nucleus in the leiomyoma (E) compared to myometrium (F). Round black dots indicate localization of protein. Black triangle points to nucleus. Cyt=cytoplasm; ECM=extracellular matrix. Magnification=21,000X. Samples from patients 18–21 were used for immunogold experiments. 4 G, H: Immunogold staining from another matched pair of leiomyoma (G) and myometrial (H) tissues stained with antisera directed against AKAP13. In this view, arrows point to AKAP13 expression in the nucleus, nuclear envelope and cytoplasm. Magnification=15,500X. Results were repeated in matched samples from 4 patients.

Figure 4. Subcellular distribution of AKAP13 in matched leiomyoma and myometrial specimens

4 A–D: Immunohistochemical localization of AKAP1 (1:500) in leiomyoma (A) and myometrium (B) tissues. Magnification=63X. Positive control, breast tissue (C); negative control=fibroid tissue stained with pre-immune antisera (D). Staining for AKAP13 was often appeared increased with a peri-nuclear appearance in fibroids. 4 E, F: Immunogold study of matched leiomyoma (E) and myometrium (F) specimens using 2665 anti-sera directed against AKAP13. Note the angular cell shape, reduced cytoplasm, and notched nucleus in the leiomyoma (E) compared to myometrium (F). Round black dots indicate localization of protein. Black triangle points to nucleus. Cyt=cytoplasm; ECM=extracellular matrix. Magnification=21,000X. Samples from patients 18–21 were used for immunogold experiments. 4 G, H: Immunogold staining from another matched pair of leiomyoma (G) and myometrial (H) tissues stained with antisera directed against AKAP13. In this view, arrows point to AKAP13 expression in the nucleus, nuclear envelope and cytoplasm. Magnification=15,500X. Results were repeated in matched samples from 4 patients.

Figure 5. Altered focalization of AKAP13 to cytoskeletal structures in immortalized cultured leiomyoma and myometrial cell lines

5 A: Confocal laser microscopy of leiomyoma cell stained with antisera directed against AKAP13 (green) and alpha smooth muscle actin (red). AKAP13 protein is localized to cytoskeletal filaments. DAPI-stained nucleus appears blue. 5 B: Confocal laser microscopy of myometrial cell stained for AKAP13 (green) and alpha smooth muscle actin (red). Staining for AKAP13 protein is less robust. DAPI-stained nucleus appears blue. Results were repeated in 3 independent experiments.

Figure 6. Evidence of activation of solid state signaling in leiomyoma cells

6A: Western analysis of phosphorylated p38MAPK in leiomyoma (L) and matched myometrial (M) samples. Findings were replicated in 3 experiments. 6 B: As a positive control for detection of phosphor-p38MAPK, lysates were prepared from Cos-7 cells were treated with anisomycin (+). Untreated Cos-7 cells served as a negative control. 6 C, D: Immunohistochemical staining of leiomyoma (C) or matched myometrial section (D) with antisera directed against phosphor-p38MAPK. Magnification=40X.

Figure 7. Model of altered mechanical stress in leiomyoma cells

Leiomyoma cells are under increased mechanical stress compared to myometrial cells. The extracellular matrix is abnormal in content and structure. Mechanical load is associated with alteration in actin organization as well as Rho-GEF expression (AKAP13). There is evidence for activation of stress-activated kinases, such as p38MAPK (pp38).