We have developed several recombinant fibronectin proteins that couple the “open” conformation of FNIII1 with integrin-binding sequences in order to mimic the effects of the ECM form of fibronectin on cell growth and migration. We compared the bioactivity of the fibronectin matrix mimetics to full-length, plasma fibronectin and to integrin-binding fragments and peptides. Our studies show that a chimeric fibronectin fragment composed of FNIII1H and FNIII8, with the RGDS loop inserted into FNIII8, supports cell adhesion and migration, stimulates collagen gel reorganization, and displays enhanced proliferative activity over integrin-binding fibronectin fragments and other full-length ECM proteins. We localized the growth-promoting activity in FNIII1H to amino acids Arg613, Arg615, and Lys617, and present new evidence that FNIII8 has cell growth- and migration-promoting properties.
Surfaces presenting the RGD-loop in FNIII8 exhibited identical adhesive activities compared to the original matrix mimetic (GST/III1H,8-10; ), which was comparable to that observed with integrin-binding fragments (). Deleting FNIII9 from the original matrix mimetic did not decrease the proliferation response of cells () and was not required for cell migration (), indicating that binding of the PHSRN sequence in FNIII9 to α5β1 integrins [43
] does not contribute to the observed activities. Surfaces coated with the chimeric RGD-construct produced higher levels of cell growth over surfaces coated with linear GRGDSP peptides (), suggesting that the structural framework of the FNIII module provides support for the RGD loop to allow for higher affinity interactions with integrin receptors [47
]. The growth-promoting activity of the chimeric fibronectin matrix mimetic, GST/III1H,8RGD
, was comparable to that of the ECM protein, vitronectin, and was greater than that observed for other full-length ECM substrates including fibrinogen and gelatin (). Thus, tethering or incorporating chimeric fibronectin matrix mimetics into biomaterials could provide robust proliferative activity to tissue-engineered scaffolds and medical devices.
The bioactivities of the fibronectin fragments tested were not identical. Surfaces coated with either GST/III1H,8-10 or GST/III1H,8,10 induced high-growth and high-migration responses; GST/III1H,8RGD
-coated surfaces provided high-growth and moderate-migration activities; GST/III8-10 induced low-growth but high-migration activities. Hence, engineering interfaces with different fibronectin matrix mimetics and fragments may represent a strategy to spatially pattern different cellular responses. In contrast to results obtained in the growth studies, a large decrease in cell migration rate occurred when FNIII10 was removed and replaced by the RGD loop (compare with ), suggesting that an auxiliary site in FNIII10 [47
] may contribute to enhanced cell migration, but not to enhanced cell growth. On the other hand, deletion of FNIII8 from GST/III1H,8-10 reduced both growth () and migration (). Previous studies have identified a region within the amino-terminus of FNIII8 that is involved in cell adhesion [48
]. Taken together, the results suggest that at least two additional sites within the cell-binding domain of fibronectin, one in FNIII10 and one in FNIII8, contribute to integrin-mediated cellular responses.
Proteolysis of the first type III repeat of fibronectin at residue Ile597
removes both the A and B β strands and results in a carboxyl-terminal fragment that binds to heparin [49
]. Our previous studies localized the growth-promoting activity of GST/III1H,8-10 to the heparin-binding sequence, K611
. Here, we demonstrate that FNIII1H is also required for the enhanced growth response to surfaces coated with GST/III1H,8-10 and further, we pinpoint the growth-enhancing sequence in FNIII1 to R613
, and K617
. The cell surface receptor that binds to FNIII1H is currently unknown. Our previous work suggests that FNIII1 interacts with cells via heparan sulfate proteoglycans [32
]. Aortic smooth muscle cells adhere to surfaces coated with micromolar concentrations of a similar FNIII1 fragment, III1-C, via a heparin-dependent mechanism that involves α5β1 integrins [50
]. Together, these studies suggest that FNIII1H may bind either directly or indirectly to α5β1 integrins via heparan sulfate proteoglycans to elicit cellular responses. In support of this, we have shown that FNIII1H must be directly coupled to FNIII8-10 to stimulate cell growth, as individual fragments added simultaneously to cells do not have growth-promoting effects [32
]. Similarly, the proliferative response to surfaces coated with a mixture of FNIII1H and FNIII8-10 fragments was not different from that observed with GST/III8-10-coated surfaces (data not shown).
Fibronectin type III repeats are found in ~2% of all human proteins and show a high degree of structural similarity [51
]. The anti-parallel β-sheets of FNIII repeats are composed of three (A, B, and E) and four (C, D, F, and G) β strands that overlap to form a hydrophobic core [3
], imparting a high degree of stability to the protein’s structure [52
]. The six loops formed at the poles between β strands (termed AB, BC, etc.) are highly variable and can withstand extensive modification without loss of stability [53
]. This inherent stability likely facilitated the insertion of the RGDS sequence into the FG loop of FNIII8. The stability of fibronectin type III repeats has also allowed for the development of FNIII-domains as scaffolds for the display of binding elements [55
], while an engineered FNIII scaffold is currently in Phase II trials as an anti-angiogenesis agent [51
]. Both FNIII1 and FNIII8 lack cysteine residues and post-translation modifications [2
], which facilitates production in bacteria. For our studies, GST/III1H,8RGD
was generated with a cleavable GST-tag that allows for rapid purification via glutathione chromatography; we obtained yields of ~ 4 mg of purified GST/III1H,8RGD
per liter of bacterial culture. The molecular mass of the chimeric matrix mimetic, excluding the GST-tag, is ~19 kDa. In contrast, the molecular mass of a single, soluble fibronectin molecule is ~500 kDa. The small size of the fibronectin matrix mimetic decreases potential immunogenicity, provides increased structure and stability over peptides, and reduces the number of binding sites for other receptors.