AbstractThe oncofetal protein Migration Stimulating Factor (MSF) is a truncated isoform of human fibronectin which exhibits numerous bioactivities that are pertinent to cancer progression. The MSF protein (70kDa) has potent motogenic activity, with only femtomolar concentrations required to produce half-maximal. The proteolytic degradation of MSF generates the functionally equivalent 43kDa Gel-BD domain and 21kDa IGD peptide. The screening of conditioned medium (CM) for bioactivity revealed two sources of MSF-inhibitory (MSF-I) activity; the spontaneously immortalised human keratinocyte cell line (HaCaT) and endothelial cells (ENDO 742) specifically when exhibiting a cobblestone phenotype.
The CM from the HaCaT keratinocyte line was fractionated by both molecular weight and ionic charge, followed by sequence analysis which identified the inhibitor as Neutrophil Gelatinase Associated Lipocalin (NGAL). Both recombinant and cell-produced NGAL neutralise the motogenic activity of MSF. This novel bioactivity for NGAL is not dependent on its iron transportation capability or direct binding to MSF. HaCaT cells also secrete MSF; the bioactivity of which is masked by the co-expression of NGAL.
The relative expression levels of the pro- and anti-motogenic factors, MSF and NGAL, were assessed using an in vitro model for human skin carcinogenesis, the HaCaT –ras clones. The shift in tumorigenic potential from benign to metastatic was characterised by a decrease in NGAL and an increase in MSF expression, indicating their potential role in tumour progression.
The protein responsible for the MSF inhibitory activity is cell- type specific; NGAL is not expressed by endothelial cells in vitro. MSF stimulates the generation of sprouting endothelial cells from a cobblestone monolayer and acts a survival factor for spontaneously sprouting cells within a 3D matrix. NGAL does not selectively the target sprouting phenotype of endothelial cells, but induces apoptosis in all endothelial cells. Fractionation of endothelial CM revealed that both sprouting and cobblestone cells express bioactive MSF and a MSF-I. Endothelial MSF-I was located in fractions of MW 70kDa, 40kDa and =25kDa; further investigation is required to identify the protein responsible.
|Date of Award||2013|
|Supervisor||Sarah Jones (Supervisor) & Dorothy Crouch (Supervisor)|
- Migration Stimulating Factor
- MSF (Migration Stimulating Factor)
- Neurophil Gelatinase Associated Lipocalin