Supramolecular Nanofibers Enhance Growth Factor Signaling by Increasing Lipid Raft Mobility

The nanostructures of self-assembling biomaterials have been previously designed to tune the release of growth factors in order to optimize biological repair and regeneration. We report here on the discovery that weakly cohesive peptide nanostructures in terms of intermolecular hydrogen bonding, when combined with low concentrations of osteogenic growth factor, enhance both BMP-2 and Wnt mediated signaling in myoblasts and bone marrow stromal cells, respectively. Conversely, analogous nanostructures with enhanced levels of internal hydrogen bonding and cohesion lead to an overall reduction in BMP-2 signaling. We propose that the mechanism for enhanced growth factor signaling by the nanostructures is related to their ability to increase diffusion within membrane lipid rafts. The phenomenon reported here could lead to new nanomedicine strategies to mediate growth factor signaling for translational targets

LS8 cells express the Eda-A1 receptor, Edar.

<p>Real-time PCR <b>(A)</b> and RT-PCR <b>(B)</b> were used to detect the expression of <i>Edar</i> in LS8 cells.</p

Schematic diagram depicting the distribution of major pH regulators in maturation-stage ameloblasts.

<p>Cftr, Slc26a1, Slc26a4, Slc26a6 and Slc26a7 are localized to the apical membrane and Slc26a1, Slc26a6 and Slc26a7 physically interact with Cftr to form regulation complexes. Slc26a7 is also found on the endo-lysosomal membrane. Ae2, Nhe1 and NBCe1 are localized basolaterally. Clcn7 is expressed on the endo-lysosomal membrane. CA2 exhibits intracellular distribution whereas CA6 functions in extracellular enamel matrix. Lamp1 in this image was used as a marker of late lysosomes.</p

Expression and receptor binding of wild-type and mutant EDA2 proteins.

<p>Representative western blots depict the expression level of FLAG-tagged wild-type and mutated EDA2 in <b>(A)</b> cell lysate and <b>(B)</b> supernatant of transfected 293T cells. <b>(C)</b> Ligands in supernatants were immunoprecipitated with XEDAR:Fc and analyzed by western blot. Precipitated XEDAR:Fc is shown in the bottom panel. <b>(D)</b> Semi-quantitative analysis of the western blots image in Fig 2C upper panel. *, <i>P</i><0.05; **, <i>P</i><0.01; WB, western blot; IP, immunoprecipitated; WT, wild-type.</p

CD spectra of mouse and chimeric amelogenins at pH 8.

<p>No difference spectrum was observed between the chimeric protein (rM180 containing <i>L. catta</i> repeats) and the wild type rp(H)M180.</p

Average of nanosphere radii in rp(H)M180 and chimeric proteins.

<p>The difference in radii is significant using student's t-test (samples were normally distributed).</p

ClustalW alignments for the <i>Lemur catta</i> and <i>Mus musculus</i> X-derived amelogenin protein, and the recombinant proteins used to study the role of the MQP repeats sequences in exon 6.

<p>Previously, the mouse cDNA backbone sequence was used to create a recombinant protein [rp(H)M180] identified here as mAmelx. Using rp(H)M180 as template DNA, a PCR-based strategy was used to create a recombinant chimeric protein that had four MQP repeats added at the region shown (bottom line). We refer to this as the mouse-lemur chimeric protein. It should be noted that the alignments presented here are based on using ClustalW version 2.1 in MacVector version11.0.4 software aligning the <i>Lemur catta</i> sequence (EU168853) and that of <i>Mus musculus</i> (NP_033796). The resulting aligned sequence of these two species differs from those reported previously which used a different alignment algorithm and software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018028#pone.0018028-Delgado1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018028#pone.0018028-Sire1" target="_blank">[22]</a>. However, this difference does not affect the analyses performed in this study of the purified proteins.</p

Thermal unfolding-refolding behavior of mouse (A) and chimeric (B) amelogenins.

<p>The figure represents change in ellipticity at 224 nm as a function of temperature. The heating and cooling cycles are represented by red and blue open circles, respectively. Note that the –MQP- inserted chimeric amelogenin undergoes similar biphasic transitions as the mouse amelogenin. The onsets of unfolding and refolding are indicated by red and blue arrows, respectively.</p

Expression and receptor binding of wild-type and mutant EDA1 proteins.

<p>Representative western blots depict the expression level of FLAG-tagged wild-type and mutated EDA1 in <b>(A)</b> cell lysate and <b>(B)</b> supernatant of transfected 293T cells. <b>(C)</b> Ligands in supernatants were immunoprecipitated with EDAR:Fc and analyzed by western blot. Precipitated EDAR:Fc is shown in the bottom panel. <b>(D)</b> Semi-quantitative analysis of the western blots image in Fig 1C upper panel. *, <i>P</i><0.05; **, <i>P</i><0.01; WB, western blot; IP, immunoprecipitated; WT, wild-type.</p

Micro-CT analysis of Slc26a1^-/- and Slc26a7^-/- mandibles.

<p>The mandibles from wild-type, <i>Slc26a1</i> null and <i>Slc26a7</i> null animals (at 8 weeks of age) were subject to micro-CT analysis (n = 3). The relative density and thickness of enamel on the labial incisor where the cortical bone enclosing just begins (A1-C2) were measured. There was no statistical difference between mutant and wild-type animals with respect to these two parameters (D-E). (Enamel Em, Dentin De)</p