As published in Journal of the Society of Gynecological Investigation, Vol. 5, No. 1, pp.44-47, 1998
Elizabeth A. Stewart, MD, Alan R. Rhoades,
BS, and Romana A. Nowak, PhD
From the Department of Obstetrics, Gynecology and Reproductive Biology,
Harvard Medical School, and Brigham and Women's Hospital, Boston, Massachusetts
OBJECTIVE:
To evaluate the levels of mRNA for the extracellular matrix proteins collagen
type I, collagen type III, and fibronectin in leiomyomas and myometrium obtained
from women treated with GnRH-agonist (GnRH-a) and to examine steroid hormone
regulation of these proteins using an in vitro explant culture system.
METHODS: Northern blot analysis of mRNA was
obtained from hysterectomy specimens at the time of surgery or after 48 hours of
in vitro steroid treatment. A portion of the tissue was processed for RNA, and
the remaining tissue was used to establish explant cultures. Extracellular
matrix/alpha-tubulin ratio was computed for each band, and within each
experiment the lowest ratio was standardized to a value of one densitometry unit
to allow for comparison among experiments.
RESULTS: There is a relative overexpression of both collagen type I and
collagen type III mRNAs but not fibronectin in leiomyomas compared to myometrium
from the same uteri of women treated with GnRH-a.
CONCLUSION: Leiomyomas obtained from women treated with GnRH-a show an
up-regulation of collagens type I and type III similar to that seen in
leiomyomas obtained from women in the proliferative phase of their menstrual
cycles.
Figure 1.Northern blot analysis of mRNA for the ECM proteins collagen type I (alpha1), collagen type III (alpha1), fibronectin, and alpha-tubulin from ten patients undergoing hysterectomy after pre-surgical treatment with 3 months of leuprolide acetate treatment. From each patient mRNA was obtained from an intramural leiomyoma (L) and adjacent normal myometrium (M) for comparison. For collagen types I and III, two transcripts were seen in most samples (5.8 and 4.8 kb and 5.4 and 4.8 kb, respectively), wheras a single transcript was seen for fibronectin (7.9 kb) and alpha-tubulin (2.2 kb). After normalization to alpha-tubulin, levels of mRNA for both collagen type I and collagen type III were found to be significantly elevated in leiomyomas compared with autologous myometrium (P < .02 for both).
Figure 2.Northern blot analysis for collagen type I (alpha1), collagen type III (alpha1), fibronectin, and alpha-tubulin from three patients after presurgical leuprolide acetate treatment where myometrium (M) and multiple leiomyomas (L) were available for study. The number after each letter L indicates the diameter of the leiomyoma in centimeters. For the 15-cm leiomyoma, the first sample was obtained from the periphery of the leiomyoma, and the second was obtained from the central portion. There was no variation in expression of any of the ECM proteins with size of the leiomyoma.
Figure 3. In vitro steroid regulation of collagen type I and collagen
type III in leiomyoma (A and C) and myometrial (B and D) tissue explants
obtained from patients who had undergone surgical pretreatment with leuprolide
acetate. All data represent treatment for 48 hours with no steroid hormones (C),
10-8 mmol/L estradiol (E), progesterone (P), or both (E + P). Each
bar represents the mean ąSEM for three experiments. After 48 hours
in culture, increased expression of both collagens is seen in leiomyomas
compared with myometrium from the same patients. There was no significant
suppression seen with any hormone treatment.