A study of the biology of perlecan

Extracellular matrix (ECM) is a component of a variety of organisms that provides both structural support and influence upon the cells it surrounds. The importance of the ECM is becoming more apparent as matrix defects are linked to human disease. In this study, the large, extracellular matrix heparan sulfate proteoglycan, perlecan (Pln) is examined in two systems. First, the role of Pln in the interaction between a blastocyst and uterine epithelial cells is investigated. In mice, blastocyst attachment and implantation occurs at approximately d 4.5 post coitus. In addition, a delayed implantation model has been used to distinguish between the response of the blastocyst to that of hatching and of becoming attachment competent. ^ The second series of experiments described in this study focuses on the process of chondrogenesis in mice. Pln, commonly expressed with other basement membrane (BM) proteins, was found to be expressed in cartilaginous tissue without other BM proteins. This unusual expression pattern led to further study and the development of an in vitro chondrogenesis assay using the mouse embryonic fibroblast cell line, C3H/10T1/2. When cultured on Pln in vitro, these cells form aggregates and express the cartilage proteins, collagen type II and aggrecan. In examining the participation of the heparan sulfate (HS) chains in this process, the proteoglycan was enzymatically digested to remove the HS chains before the initiation of 10T1/2 cell culture. After digestion, the ability of Pln to stimulate aggregate formation was greatly diminished. Thus, the HS chains participate in the cell induction process. To determine which domain of Pln might be responsible for this activity, recombinant fragments of Pin were used in the cell culture assay. Of all recombinant protein fragments tested, only the domain including the HS chains, domain 1, was able to initiate the morphological change exhibited by the 10T1/2 cells. Similar to native Pln, when HS chains were removed from domain I, chondrogenic activity was abolished. A variant of domain I carrying both HS and chondroitin sulfate (CS) chains retained activity when only HS chains were removed. When both HS and CS chains were removed, then activity was lost. ^ The ability to rapidly stimulate differentiation of 10T1/2 cells in vitro may lead to better control of chondrogenesis in vitro and in vivo, providing better understanding and manipulation of the chondrogenic process. This greater understanding may have benefits for study of cartilage and bone diseases and subsequent treatment options. (Abstract shortened by UMI.)^

THE EFFECT OF RELAXIN ON BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS OF RAT MYOMETRIAL CELLS IN CULTURE (CYCLIC-AMP, CELL SHAPE CHANGE)

Relaxin is able to inhibit spontaneous, oxytocin-and prostaglandin-driven uterine contractions. The intracellular mechanism of action of relaxin on uterine relaxation had previously been studied using isometrically suspended uterine strips. Since uterine strips contain stroma as well as myometrium, the changes in biochemical parameters induced by relaxin treatment may not occur in the same cell types responsible for the physical changes. In these studies, cultures of enriched populations of rat myometrial cells were used to investigate the effect of relaxin on biochemical and morphological parameters which are related to relaxation.^ Under optimal culture conditions (initial plating density 1 - 1.5 x 10('6)cells/ml, 3 ml/35 mm dish, 2 days culture), enzymatically isolated rat myometrial cells were able to respond to relaxin with cAMP elevation. Relaxin elevated cAMP levels in the presence but not the absence of 0.1 mM methylisobutylxanthine or 0.4 um forskolin in a time- and concentration-dependent manner. In contrast, isoproterenol was able to elevate cAMP levels in the presence and absence of 0.1 mM methylisobutylxanthine.^ Oxytocin treatment caused a decrease in mean cell length and area of myometrial cells in culture which could be considered analogous to contraction. Under optimal culture conditions, relaxin increased myometrial cell length and area (i.e. analogous to relaxation) of oxytocin-treated cells in a time- and concentration-dependent manner. Other relaxants such as isoproterenol and dibutyryl cAMP also increased cell length and area of oxytocin - treated myometrial cells in culture.^ Under optimal culture conditions, relaxin decreased myosin light chain kinase activity in a time-and concentration-dependent manner by increasing the K(,50) of the enzyme for calmodulin (CaM), i.e. decreasing the affinity of the enzyme for CaM. The decrease in the affinity of myosin light chain kinase for CaM may be due to the phosphorylation of the enzyme by cAMP-dependent protein kinase. Relaxin also decreased the Ca('2+)(.)CaM-independent myosin light chain kinase activity to a lesser extent than that of the Ca('2+)(.)CaM-dependent enzyme activity. This was not attributable to a decrease in the affinity of the enzyme for myosin in myometrial cells in culture, in contrast to the finding of such a change following relaxin treatment of uterine strips. Further studies are required to clarify this point.^ There was a temporal association between the effects of relaxin on elevation of cAMP levels in the presence of 0.4 uM forskolin, increase in cell length and decrease in myosin light chain kinase activity. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^