Search

BACKGROUND: Matrix metalloproteinases (MMPs) play key roles in remodeling of the extracellular matrix during embryogenesis and fetal development. The objective of this study was to determine the effects of prinomastat, a potent selective MMP inhibitor, on fetal growth and development. METHODS: Prinomastat (25, 100, 250 mg/kg/day, p.o.) was administered to pregnant female Sprague–Dawley rats on gestational days (GD) 6–17. A Cesarian section was carried out on GD 20 and the fetuses were evaluated for viability and skeletal and soft tissue abnormalities. RESULTS: Prinomastat treatment at the 250 mg/kg/day dose produced a decrease in body weight and food consumption in the dams. A dose‐dependent increase in post‐implantation loss was observed in the 100 and 250 mg/kg/day‐dose groups, resulting in only 22% of the dams having viable litters for evaluation at the 250 mg/kg/day dose. Fetal skeletal tissue variations and malformations were present in all prinomastat treated groups and their frequency increased with dose. Variations and malformation in fetal soft tissue were also increased at the 100 and 250 mg/kg/day doses. Prinomastat also interfered with fetal growth of rat embryo cultures in vitro. CONCLUSIONS: These data confirm that MMP inhibition has a profound effect on fetal growth and development in vivo and in vitro. Birth Defects Research (Part B) 77:95–103, 2006. © 2006 Wiley‐Liss, Inc.

Culture models of target cells are anticipated to help elucidate the mechanism by which inorganic arsenic acts as a carcinogen in humans. Present work characterizes the response of human keratinocytes, a target cell type, to arsenic suppression of their differentiation program. Four representative differentiation marker mRNAs (involucrin, keratinocyte transglutaminase, small proline-rich protein 1, and filaggrin) were suppressed by both arsenate and arsenite in normal, spontaneously immortalized (premalignant), and malignant keratinocytes with EC50 values in the low micromolar range. The suppression was almost completely reversed 9 days after removal of arsenate from the culture medium. In the case of the involucrin gene, suppression was mediated primarily by two functional AP1 response elements in the gene promoter. Both glucocorticoid and serum stimulation of differentiation occurred to a similar extent in the presence and absence of arsenic, indicating neither stimulation was a specific target of arsenic action and neither agent could overcome arsenic suppression. In contrast, 12-O-tetradecanoylphorbol-13-acetate prevented the suppression of keratinocyte transglutaminase, suggesting that arsenic acts upstream of protein kinase C.

Keratinocyte transglutaminase catalyzes isopeptide bond formation to yield the highly insoluble cross-linked envelope during terminal differentiation of epidermal cells. Transcriptional response elements were identified in the 5′-flanking DNA of the gene for this enzyme by a combination of transient transfection and electrophoretic mobility shift analyses. Since human keratinocytes transcribed ineffectively transfected transglutaminase flanking DNA, a key feature of these experiments was the use of rat bladder epithelial cells as recipients. Serial deletion experiments identified by transient transfection an important response region containing three putative AP2-like response elements approximately 0.5 kilobases from the transcription initiation site. Oligonucleotides, each containing a single one of the elements, formed specific complexes with keratinocyte nuclear proteins. Two of the response elements were found to be functional by transfection in site-specific deletion experiments. Of these one formed specific DNA-protein complexes with nuclear proteins only from cells exhibiting keratinocyte differentiation. UV cross-linking experiments estimated the protein component of the complex to be ≈85 kDa. This response element alone increased substantially the transcription of a minimal transglutaminase promoter in transient transfections. Further characterization of the putative transcription factor binding to this response element may provide insight into the regulation of keratinocyte transglutaminase.