Engineering topics

Partridge, Nicola C.

Publications and source records attributed to Partridge, Nicola C..

5 records

NASA NTRS2002

Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

NASA Discipline Musculoskeletal↗

NASA NTRS2002

Parathyroid hormone-dependent signaling pathways regulating genes in bone cells

Parathyroid hormone (PTH) is an 84-amino-acid polypeptide hormone functioning as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. PTH and PTH-related protein (PTHrP) indirectly activate osteoclasts resulting in increased bone resorption. During this process, PTH changes the phenotype of the osteoblast from a cell involved in bone formation to one directing bone resorption. In addition to these catabolic effects, PTH has been demonstrated to be an anabolic factor in skeletal tissue and in vitro. As a result, PTH has potential medical application to the treatment of osteoporosis, since intermittent administration of PTH stimulates bone formation. Activation of osteoblasts by PTH results in expression of genes important for the degradation of the extracellular matrix, production of growth factors, and stimulation and recruitment of osteoclasts. The ability of PTH to drive changes in gene expression is dependent upon activation of transcription factors such as the activator protein-1 family, RUNX2, and cAMP response element binding protein (CREB). Much of the regulation of these processes by PTH is protein kinase A (PKA)-dependent. However, while PKA is linked to many of the changes in gene expression directed by PTH, PKA activation has been shown to inhibit mitogen-activated protein kinase (MAPK) and proliferation of osteoblasts. It is now known that stimulation of MAPK and proliferation by PTH at low concentrations is protein kinase C (PKC)-dependent in both osteoblastic and kidney cells. Furthermore, PTH has been demonstrated to regulate components of the cell cycle. However, whether this regulation requires PKC and/or extracellular signal-regulated kinases or whether PTH is able to stimulate other components of the cell cycle is unknown. It is possible that stimulation of this signaling pathway by PTH mediates a unique pattern of gene expression resulting in proliferation in osteoblastic and kidney cells; however, specific examples of this are still unknown. This review will focus on what is known about PTH-mediated cell signaling, and discuss the established or putative PTH-regulated pattern of gene expression in osteoblastic cells following treatment with catabolic (high) or anabolic (low) concentrations of the hormone.

NASA Discipline Musculoskeletal↗

NASA NTRS2002

Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation

Previously, we determined that the activator protein-1 (AP-1)-binding site and the runt domain (RD)-binding site and their binding proteins, c-Fos.c-Jun and Cbfa, regulate the collagenase-3 promoter in parathyroid hormone-treated and differentiating osteoblasts. Here we show that Cbfa1 and c-Fos.c-Jun appear to cooperatively bind the RD- and AP-1-binding sites and form ternary structures in vitro. Both in vitro and in vivo co-immunoprecipitation and yeast two-hybrid studies further demonstrate interaction between Cbfa1 with c-Fos and c-Jun in the absence of phosphorylation and without binding to DNA. Additionally, only the runt domain of Cbfa1 was required for interaction with c-Jun and c-Fos. In mammalian cells, overexpression of Cbfa1 enhanced c-Jun activation of AP-1-binding site promoter activity, demonstrating functional interaction. Finally, insertion of base pairs that disrupted the helical phasing between the AP-1- and RD-binding sites also inhibited collagenase-3 promoter activation. Thus, we provide direct evidence that Cbfa1 and c-Fos.c-Jun physically interact and cooperatively bind the AP-1- and RD-binding sites in the collagenase-3 promoter. Moreover, the AP-1- and RD-binding sites appear to be organized in a specific required helical arrangement that facilitates transcription factor interaction and enables promoter activation.

Non-NASA Center↗

NASA NTRS1997

Skeletal Collagen Turnover by the Osteoblast

Among the most overt negative changes experienced by man and experimental animals under conditions of weightlessness are the loss of skeletal mass and attendant hypercalciuria. These clearly result from some disruption in the balance between bone formation and bone resorption (i.e. remodelling) which appears to be due to a decrease in the functions of the osteoblast. In the studies funded by this project, the clonal osteoblastic cell line, UMR 106-01, has been used to investigate the regulation of collagenase and Tissue Inhibitors of MetalloProteases (TIMPs). This project has shed light on the comprehensive role of the osteoblast in the remodelling process, and, in so doing, provided some insight into how the process might be disrupted under conditions of microgravity.

Partridge, Nicola C.↗

NASA NTRS1993

Bone culture research

The experiments described are aimed at exploring PTH regulation of production of collagenase and protein inhibitors of collagenase (tissue inhibitors of metalloproteases, TIMP-1 and -2) by osteoblast-like osteosarcoma cells under conditions of weightlessness. The results of this work will contribute to information as to whether a microgravity environment alters the functions and responsiveness of the osteoblast. The objectives of the Bone Culture Research (BCR) experiment are: to observe the effects of microgravity on the morphology, rate of proliferation, and behavior of the osteoblastic cells, UMR 106-01; to determine whether microgravy affects the hormonal sensitivity of osteroblastic cells; and to measure the secretion of collagenase and its inhibitors into the medium under conditions of microgravity. The methods employed will consist of the following: the osteoblast-like cells, UMR-106-01, will be cultured in four NASDA cell culture chambers; two chambers will be subjected to microgravity on SL-J; two chambers will remain on the ground at KSC as ground controls but subjected to an identical set of culture conditions as on the shuttle; media will be changed four times; twice the cells will receive the hormone parathyroid hormone-related protein (PTHrP) and media collected; cells will be photographed under conditions of microgravity; and media and photographs will be analyzed upon return to determine whether functions of the cells changed.

Partridge, Nicola C.↗