Proteoglycans in Leiomyoma and Normal Myometrium: Abundance, Steroid Hormone Control, and Implications for Pathophysiology.

Uterine leiomyoma are a common benign pelvic tumors composed of modified smooth muscle cells and a large amount of extracellular matrix (ECM). The proteoglycan composition of the leiomyoma ECM is thought to affect pathophysiology of the disease. To test this hypothesis, we examined the abundance (by immunoblotting) and expression (by quantitative real-time polymerase chain reaction) of the proteoglycans biglycan, decorin, and versican in leiomyoma and normal myometrium and determined whether expression is affected by steroid hormones and menstrual phase. Leiomyoma and normal myometrium were collected from women (n = 17) undergoing hysterectomy or myomectomy. In vitro studies were performed on immortalized leiomyoma (UtLM) and normal myometrial (hTERT-HM) cells with and without exposure to estradiol and progesterone. In leiomyoma tissue, abundance of decorin messenger RNA (mRNA) and protein were 2.6-fold and 1.4-fold lower, respectively, compared with normal myometrium. Abundance of versican mRNA was not different between matched samples, whereas versican protein was increased 1.8-fold in leiomyoma compared with myometrium. Decorin mRNA was 2.4-fold lower in secretory phase leiomyoma compared with proliferative phase tissue. In UtLM cells, progesterone decreased the abundance of decorin mRNA by 1.3-fold. Lower decorin expression in leiomyoma compared with myometrium may contribute to disease growth and progression. As decorin inhibits the activity of specific growth factors, its reduced level in the leiomyoma cell microenvironment may promote cell proliferation and ECM deposition. Our data suggest that decorin expression in leiomyoma is inhibited by progesterone, which may be a mechanism by which the ovarian steroids affect leiomyoma growth and disease progression.

Proteoglycans of uterine fibroids and keloid scars: similarity in their proteoglycan composition.

Fibrosis is the formation of excess and abnormal fibrous connective tissue as a result of either a reparative or reactive process. A defining feature of connective tissue is its extracellular matrix, which provides structural support and also influences cellular activity. Two common human conditions that result from fibrosis are uterine fibroids (leiomyomas) and keloid scars. Because these conditions share a number of similarities and because their growth is due primarily to excessive extracellular matrix deposition, we compared the proteoglycans of uterine fibroids and keloid scars with corresponding normal tissues. Our analysis indicates that uterine fibroids and keloid scars contain higher amounts of glycosaminoglycans relative to normal myometrium and normal adult skin respectively. Proteoglycan composition is also different in the fibrotic tissues. Compared with unaffected tissues, uterine fibroids and keloid scars contain higher relative amounts of versican and lower relative amounts of decorin. There is also evidence for a higher level of versican catabolism in the fibrotic tissues compared with unaffected tissues. These qualitative and quantitative proteoglycan differences may play a role in the expansion of these fibroses and in their excessive matrix deposition and matrix disorganization, due to effects on cell proliferation, TGF (transforming growth factor)-ß signalling and/or collagen fibril formation.

Pericellular versican regulates the fibroblast-myofibroblast transition: a role for ADAMTS5 protease-mediated proteolysis.

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5(-/-) mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFß signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5(-/-) cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcan(hdf/+)) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5(-/-) cells resulted from increased PCM versican content, we generated Adamts5(-/-);Vcan(hdf/+) mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5(-/-) mice. In Adamts5(-/-) fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates.

Production of a monoclonal antibody, DF-5, that identifies cells at the epithelial-mesenchymal interface in normal human skin. APN/CD13 is an epithelial-mesenchymal marker in skin.

Epithelial-mesenchymal interactions play a critical role in skin development and differentiation, and similar interactions may also regulate the day-to-day proliferation and differentiation events of the epidermis that occur in normal adult skin. This study was directed at identifying molecules that are selectively located at the dermal-epidermal junction in normal adult skin as they may be involved in regulating these homeostatic events. To this end, monoclonal antibodies were raised against the crude cell membrane fraction of cultured human dermal fibroblasts. Screening of antibodies that recognized cell surface antigen on cultured human dermal fibroblasts was followed by determining which of these antibodies selectively localized cells at sites of epithelial-mesenchymal interactions. Antibody DF-5 fit these criteria and was further characterized. This antibody was found to recognize the cell surface ectopeptidase aminopeptidase N (APN), a molecule homologous to the cluster differentiation antigen CD13. Antibody DF-5 and anti-CD13 antibodies both identified cells at sites of epithelial-mesenchymal interactions in fetal, neonatal, and adult human skin, and the APN/CD13 enzyme activity was also identified at these sites. A second ectopeptidase, dipeptidyl peptidase IV (DPPIV) or CD26, presented a significantly different immunohistochemical and histochemical pattern in skin samples, confirming the specificity of the APN/CD13 studies. The function of APN/CD13 in skin has yet to be determined. Its invariant localization at sites of epithelial-mesenchymal interactions argues for a role particular to this region. It may play a role in regulating the activity of neuropeptides or other signaling peptides that are released in this region of skin or it may have an as yet undefined role in mediating communication between dermal and epidermal cells.