We are currently conducting the following research projects:

Studies on the mechanism of protease-activated receptor-2-mediated inhibition ofosteoclast differentiation 

In collaboration with Dr R. Pike, Monash University

Protease-activated receptor-2 or PAR-2 is a member of the seven-transmembrane domain G protein-coupled receptor family, which is activated by proteolytic cleavage of its extracellular domain (Figure 1). Enzymatic activators include trypsin, mast cell tryptase and factor Xa.

We have recently demonstrated that PAR-2 is expressed by osteoblasts in vivo and in vitro, however, until recently we have been unable to identify any function for PAR-2 in these cells. 

As activation of PAR-2 results in the production of inflammatory mediators in other cell types, we have hypothesized that PAR-2 activation plays a role in osteoblast-mediated osteoclast differentiation. 

We are currently conducting osteoclast differentiation assays in mouse bone marrow cultures to test the effect of PAR-2 activation upon osteoclast differentiation. 

Studies on the role of protease -activated receptor-1 in responses of bone cells to thrombin

In collaboration with Dr R. Pike, Monash University

Protease-activated receptor -1 (PAR-1) is another member of the seven transmembrane domain G-protein coupled receptor family, and is activated by thrombin (factor IIa) 

Thrombin is known to have a potent effect upon bone resorption in vitro and we have previously demonstrated that thrombin acting through PAR-1 is able to inhibit differentiation and stimulate proliferation of osteoblasts.

This project is split into two main areas of research:- 

We have recently established a model of bone repair in mice, which we have found to be superior to any of the previously published models for this species. We are currently using this bone repair model along with PAR-1 null mice, to investigate the role played by PAR-1 during bone repair and remodelling following fracture. 

We are also studying the role played by PAR-1 in the resorption of bone by osteoclasts in vitro. As mentioned before, thrombin increases the resorption of bone in tissue culture. Our studies are designed to determine if PAR-1 activation is responsible for this effect and whether thrombin treatment of cultures (i) increases the proportion of cells forming osteoclasts, (ii) increases the activity of osteoclasts or (iii) a combination of the two. 

Investigation of PAR-1-independent responses to thrombin in bone and muscle

In collaboration with Dr R. Pike, Monash University

As well as the effects that thrombin has on osteoblast and myoblast proliferation and differentiation, we have also shown that thrombin inhibits serum deprivation-induced apoptosis of primary rat and mouse osteoblasts, myoblasts and Saos-2 cells, and dexamethasone-induced apoptosis of primary mouse osteoblasts .

Apoptosis in serum deprived cells isolated from PAR-1 null mice is inhibited to the same extent as apoptosis in wild type cells, suggesting that PAR-1 is not involved in this effect. 

Thrombin's effect on apoptosis does, however, appear to be mediated by a second secreted factor which acts in an autocrine fashion to inhibit apoptosis. 

These observations have raised a number of interesting questions about the mechanism of thrombin's effect on osteoblast and myoblast apoptosis:

  1. What is the identity of the thrombin receptor involved? 
  2. What is the identity of the secreted inhibitor of apoptosis? 
  3. What are the signalling mechanisms used by the novel receptor to effect expression of the inhibitor? 
  4. What are the intracellular events elicited by the inhibitor to inhibit apoptosis?

We are currently using a number of techniques such as 2-D gel electrophoresis, cDNA expression arrays and real time PCR to answer these questions.

Regulation of bone cell function by tenascins

We have previously demonstrated that Tenascin-C supports differentiation of cultured osteoblast-like cells. We are currently using transgenic mice to investigate:-

1) The mechanism of Tenascin-C's effect on osteoblasts

2) The role of Tenascin-C in bone healing and re-modelling

3) The hypothesis that Tenascin-C helps to mediate the osteogenic response to transient stimuli such as hormones, growth factors and mechanical load.

4) The possibility that other members of the tenascin gene family regulate bone cell function.

Pathophysiological mechanisms in equine dyschondroplasia (osteochondrosis)

Long bones of the limbs normally grow through a process of replacement of cartilage with bone. This process of ossification requires the sequential proliferation, hypertrophy and apoptosis of the chondrocytes of the articular cartilage. Dyschondroplasia is a disease which affects the growth cartilage of growing animals. The processes of chondrocyte hypertrophy and/or apoptosis appear to be disrupted in the joints of individuals affected by this disease. The result is a failure of ossification and the presence of retained cartilage in the bones of such individuals (Figure 5). The presence of this cartilage compromises the structural integrity of joints leaving them prone to fracture. As such, dyschondroplasia is important in industries such as horse racing.

The aims of this project are to:-

  1. Identify changes in gene expression associated with the initiation of dyschondroplasia. 
  2. To determine how factors present in the circulation can lead to the initiation of dyschondroplastic lesions. 
  3. To evaluate the usefulness of nuclear scintigraphy in the diagnosis of dyschondroplasia/osteochondrosis in young horses. 

Muscle hypertrophy and atrophy in commercial sheep breeds

As part of a Meat and Livestock Australia - Australian Wool Innovation funded program, we are in the process of beginning a project which aims study muscle growth and development in sheep.

Protease-activated receptors and pathogenesis in P. gingivalis

In collaboration with Dr R. Pike, Monash University

Porphyromonas gingivalis is an obligate anaerobe that is a major etiological agent of periodontal disease. The bacterium secretes a number of cysteine proteases, which are known as gingipains and are believed to be important virulence factors. 

Two of the gingipains, HRgpA and RgpB, solely hydrolyse peptide bonds after arginine residues and exist as soluble secreted forms. 

Previous work in Dr Pike's laboratory has demonstrated the ability of HRgpA and RgpB to activate protease-activated receptors including PAR-1 and PAR-2. 

The current study aims to investigate the role of bacterial proteases in eliciting cellular responses in human cells.

For all research-related enquiries, please contact:

Professor Eleanor J. Mackie, Research Group Leader
Email: ejmackie@unimelb.edu.au
Telephone: +61 3 8344 7360
Facsimile: +613 8344 7374