Composition-controlled artificial collagen shows opposing roles of collagen-binding integrins and discoidin domain receptors in neuronal differentiation of PC12 cells
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Collagen, a major component of the extracellular matrix, regulates cellular behaviors, such as adhesion, differentiation, and angiogenesis. These functions are mediated by interactions between specific amino acid motifs within the collagen triple-helical structure and collagen-binding biomolecules. These include cell-surface receptors, such as integrins, discoidin domain receptors (DDRs), and syndecans, a family of transmembrane heparan sulfate proteoglycans (HSPGs). Signals mediated by these receptors are integrated to regulate cell fate. However, native collagen simultaneously presents multiple receptor-binding motifs, making it difficult to isolate receptor-specific functions and to evaluate receptor crosstalk. Here, we introduce a composition-controlled artificial collagen matrix platform that enables independent tuning of multiple receptor-binding motifs within a constant triple-helical scaffold. This material was produced by disulfide crosslinking of chemically synthesized collagen-like triple-helical peptides, each bearing a single defined receptor-binding sequence. By varying the mixing ratios of these peptides before crosslinking, we systematically controlled the composition of receptor-binding motifs within the matrices. We applied this platform to nerve growth factor-dependent neuronal differentiation of PC12 cells, a process supported by collagen. Matrices containing only integrin-binding sequences were sufficient to support this differentiation. Incorporation of an HSPG-binding sequence had little additional effect, whereas incorporation of a DDR-binding sequence suppressed integrin-mediated differentiation and coincided with DDR phosphorylation. These results reveal opposing roles of collagen-binding integrins and DDRs in regulating PC12 cell differentiation. Composition-controlled artificial collagen provides a versatile matrix platform for dissecting functional crosstalk among collagen receptors.
Statement of significance
Native collagen presents multiple receptor-binding motifs simultaneously, making it difficult to isolate receptor-specific functions and interrogate receptor crosstalk. We address this limitation with composition-controlled artificial collagen matrices assembled from chemically synthesized collagen-like triple-helical peptides. This approach enables independent, tunable presentation of defined receptor-binding sequences within a constant triple-helical scaffold. Using nerve growth factor-treated PC12 cells as a model, an integrin-binding sequence was sufficient to induce neuronal differentiation. Notably, co-presenting a discoidin domain receptor (DDR)-binding sequence suppressed this response and coincided with DDR activation. This modular matrix strategy enables controlled interrogation of collagen receptor crosstalk and supports the design of cell-instructive biomaterials.
