Breckon, Jeremy John William; (1995) Matrix metalloproteinase and TIMP gene expression during craniofacial embryogenesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes several investigations that document the synthesis and distribution of matrix metalloproteinases (MMPs; collagenase, gelatinase and stromelysin) and their inhibitor TIMP-1 (tissue inhibitor of metalloproteinases-1) in the developing craniofacial region of the rabbit. Expression of MMP mRNAs was also investigated using in situ hybridization. MMPs and TIMP-1 were detected by indirect immunofluorescence microscopy as bright intracellular accumulations probably within Golgi vesicles and also as diffuse matrix-bound extracellular deposits. Enzymes and inhibitor were synthesized at several sites of chondrogenesis and osteogenesis during both intramembranous and endochondral ossification. Regional and temporal variations in the patterns of synthesis of MMPs and TIMP-1 were documented in the developing condylar cartilage of the craniomandibular joint. Osteogenic tissues exhibited a particularly high level of gelatinase-A synthesis in periosteal and bone cells. In addition, extensive extracellular deposits of gelatinase-A were detected in the osteoid layer of newly formed bone. Osteoclasts synthesised gelatinase-AB, suggesting they may degrade matrix during bone remodelling. MMP and TIMP-1 synthesis was also shown in early tooth germs and in chondrocytes of Meckel's cartilage during involution. Stromelysin mRNA was expressed at very low levels by cells of the perichondrium and trabecular bone. Expression was upregulated in stimulated tissues but surprisingly only occasional hypertrophic chondrocytes were positive. The pattern of expression did not correlate with the immunolocalization of protein product; the reasons for this remain obscure. Cranial fold and trunk neural tube explants from mouse embryos were used to prepare neural crest cell (NCC) cultures. Gelatinase was immunolocalized in neural tube and migrating NCCs in vitro. In addition, SDS-gelatin gel zymography detected gelatinases in supernatants from short term NCC cultures; both gelatinase-A and gelatinase-B were constitutively synthesized. Enzymes were present as latent and active forms and as complexes with TIMPs. The pattern of synthesis was modified by different substrata and upregulated by interleukin-1α.

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