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. 2017 May 1;102(5):1631-1641.
doi: 10.1210/jc.2016-3527.

Versican Proteolysis by ADAMTS Proteases and Its Influence on Sex Steroid Receptor Expression in Uterine Leiomyoma

Ndeye-Aicha Gueye  1   2 , Timothy J Mead  1 , Christopher D Koch  1   3 , Charles V Biscotti  4 , Tommaso Falcone  2 , Suneel S Apte  1
Affiliations

Versican Proteolysis by ADAMTS Proteases and Its Influence on Sex Steroid Receptor Expression in Uterine Leiomyoma

Ndeye-Aicha Gueye et al. J Clin Endocrinol Metab. .

Abstract

Context: Leiomyomas have abundant extracellular matrix (ECM), with upregulation of versican, a large proteoglycan.

Objective: We investigated ADAMTS (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs) protease-mediated versican cleavage in myometrium and leiomyoma and the effect of versican knockdown in leiomyoma cells.

Design: We used quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, immunohistochemistry, and RNA in situ hybridization for analysis of myometrium, leiomyoma and immortalized myometrium and leiomyoma cells. Short interfering RNA (siRNA) was used to knockdown versican in leiomyoma cells.

Setting: This study was performed in an academic laboratory.

Patients: Study subjects were women with symptomatic or asymptomatic leiomyoma.

Main outcome measures: We quantified messenger RNAs (mRNAs) for versican splice variants. We identified ADAMTS-cleaved versican in myometrium and leiomyoma and ADAMTS messenger RNAs and examined the effect of VCAN siRNA on smooth muscle differentiation and expression of estrogen and progesterone receptors.

Results: The women in the symptomatic group (n = 7) had larger leiomyoma (P = 0.01), heavy menstrual bleeding (P < 0.01), and lower hemoglobin levels (P = 0.02) compared with the asymptomatic group (n = 7), but were similar in age and menopausal status. Versican V0 and V1 isoforms were upregulated in the leiomyomas of symptomatic versus asymptomatic women (P = 0.03 and P = 0.04, respectively). Abundant cleaved versican was detected in leiomyoma and myometrium, as well as in myometrial and leiomyoma cell lines. ADAMTS4 (P = 0.03) and ADAMTS15 (P = 0.04) were upregulated in symptomatic leiomyomas. VCAN siRNA did not effect cell proliferation, apoptosis, or smooth muscle markers, but reduced ESR1 and PR-A expression (P = 0.001 and P = 0.002, respectively).

Conclusions: Versican in myometrium, leiomyomas and in the corresponding immortalized cells is cleaved by ADAMTS proteases. VCAN siRNA suppresses production of estrogen receptor 1 and progesterone receptor-A. These findings have implications for leiomyoma growth.

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Figures

Figure 1.
Figure 1.
Abundant VCAN mRNA expression in human myometrium and leiomyoma. (a) Hematoxylin and eosin staining shows well-organized myometrium and disordered leiomyoma tissue with abundant ECM. RNA ISH using specific probes for VCAN exon 7 (GAG-α domain) and VCAN exon 8 (GAG-β domain) demonstrates strong expression (red stain) in both myometrium and leiomyoma cells. The negative control shows ISH using a bacterial probe [for dihydrodipicolinate reductase (dapB)], where no signal is evident. (b) qRT-PCR demonstrates increased VCAN V0 (P = 0.03) mRNA expression in the myometrium of symptomatic participants. (c) Leiomyomas of the symptomatic group had a greater abundance of VCAN V0 (P = 0.03) and V1 (P = 0.04) mRNAs. Asx, asymptomatic; Sym, symptomatic. *P < 0.05. Statistical significance was calculated using Student t test.
Figure 2.
Figure 2.
Immunofluorescence with versican antibodies and versican neoepitope antibodies. (a) Schematic illustrating versican variants and their cleavage by ADAMTS proteases. Each versican variant is shown bound to hyaluronan (HA) through its G1 domain. The inclusion or exclusion of alternatively spliced CS-bearing GAG-α (blue, encoded by exon 7) and GAG-β (red, encoded by exon 8) domains defines the splice variants. CS chains are shown as wavy light blue lines. The cleavage sites in V0, V1, and V2 are indicated by black arrows. The boxed illustrations on the right show specific versican fragments generated by ADAMTS proteases [i.e., versikine (the N-terminal V1 isoform fragment detected by anti-DPEAAE) and glial hyaluronate-binding protein (GHAP; the N-terminal V0/V2 isoform fragment detected by anti-NIVSFE)]. (b) Immunofluorescence of total and cleaved versican in myometrium and leiomyoma. Versican staining is green, and DAPI nuclear staining is blue. Overall, versican staining was stronger in leiomyoma than in myometrium sections. The antibodies used are indicated above each column. [Figure 2(a) illustration by David Schumick CMI. Reprinted with the permission of the Cleveland Clinic Center for Medical Art & Photography © 2016. All Rights Reserved.]
Figure 3.
Figure 3.
Western blotting of versican in myometrium and leiomyoma tissues and expression of versican-degrading ADAMTS proteases. (a and b) Western blots of matched myometrium and leiomyoma from three symptomatic patients demonstrate a distinct 70-kDa band using anti-Vc and 70-kDa and 50-kDa bands with anti-DPEAAE. Intact versican was not seen using the anti-Vc antibody, given the absence of bands at 450 kDa (versican V0) and 350 kDa (versican V1). (c) Significantly higher expression of ADAMTS4 (P = 0.006) and ADAMTS9 (P = 0.02) mRNAs was detected in the myometrium of the symptomatic group as compared with the asymptomatic group. (d) In leiomyomas from symptomatic participants, higher levels of ADAMTS4 (P = 0.03) and ADAMTS15 (P = 0.04) mRNAs were detected. Asx, asymptomatic; Sym, symptomatic. *P < 0.05. Statistical significance was calculated using Student t test.
Figure 4.
Figure 4.
Characterization of versican and ADAMTS protease expression in UtSMC-hTERT and UtLM-hTERT cells. (a) VCAN V0 (2227-fold; P = 1.46e−05) and V1 (6450-fold; P = 1.21e−05) mRNAs were more abundant in the UtLM-hTERT cells compared with the UtSMC-hTERT cells. Although VCAN V2 (29-fold; P = 4.6e−05) and V3 (85-fold; P = 2.6e−05) mRNAs were present in both cell lines and increased in UtLM-hTERT cells, they were at much lower levels than V0 and V1. (b) ADAMTS15 mRNA (P = 0.003) was significantly higher in UtLM-hTERT cells than in UtSMC-hTERT cells. ADAMTS5 (P = 0.005) and ADAMTS9 (P = 0.002) mRNAs were significantly lower in UtLM-hTERT cells. (c and d) Western blotting with the indicated antibodies identified anti-DPEAAE and anti–Vc-reactive cleaved (arrowhead) but not full-length versican (>350 kDa). Anti–glyceraldehyde-3-phosphate dehydrogenase was used as evidence of comparable cell number.
Figure 5.
Figure 5.
VCAN siRNA transfection reduces immunoreactive versican and affects estrogen and progesterone receptor expression. (a) VCAN siRNA led to 93% reduction of VCAN mRNA in UtLM-hTERT cells. (b) Western blot with anti-Vc demonstrates effective knockdown in the UtLM-hTERT cell line. Anti–glyceraldehyde-3-phosphate dehydrogenase was used as evidence of comparable cell number. (c) The expression of ACTA2 (α-SMA; P = 0.83), TAGLN (Transgelin, Sm22-α; P = 0.34), CNN1 (Calponin-1; P = 0.44), or MYH11 mRNAs (Myosin, Heavy Chain 11, Smooth Muscle; P = 0.45) did not differ between the VCAN siRNA and the control siRNA-transfected cells (siControl). (d) Phalloidin–fluorescein isothiocyanate staining of the actin cytoskeleton and SMA immunofluorescence demonstrated no change in UtLM-hTERT morphology and cytoskeleton after VCAN knockdown. Phosphohistone-H3 staining showed comparable cell proliferation. (e) qRT-PCR shows reduced expression of ESR1 (0.54-fold; P = 0.02) and PGR-A (0.62-fold; P = 0.001) in UtLM-hTERT cells after VCAN knockdown. PGR-B was unaffected.
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