PMC

(A) Confocal immunofluorescent analysis of primary enamel organ epithelial (EOE) cells treated with ScrRGDS PA for 15 min and imaged with a specific antibody against phosphorylated c-Jun Ser63 (p-c-Jun) antibody (green) with Alexa Fluor 594 phalloidin staining for F-actin (red);
(B), primary EOE cells treated with bRGDS PA for 15 min. Compared to control PA matrix lacking signaling capacity, the primary EOE cells treated with bioactive bRGDS PA revealed increased p-c-Jun accumulation in the nuclei (arrowhead);
(C), primary EOE cells treated with bRGDS PA for 15 min in the presence of 500 nM FAK inhibitor PF 573228 revealed the reduction of nuclear localization signal for p-c-Jun;
(D) Primary enamel organ epithelial (EOE) cells were treated with either ScrRGDS PA or bioactive bRGDS PA and their lysates analyzed by Western blot analysis for expression of total c-Jun and activated phospho-c-Jun (p-c-Jun). The p-c-Jun expression was increased in primary EOE cells treated with bRGDS PA compared with ScrRGDS PA (*p<0.05, student t-test), while the levels of total c-Jun demonstrated little variation between treatment groups;
(E) Densitometry analysis of the intensity of the detected band normalized to GAPDH shown as a bar graph. The activated phospho-c-Jun protein level was approximately 1.5 times as abundant in primary EOE cells treated with bRGDS PA compared with scrambled PA control.

(A) Cellular FAK phosphorylation on tyrosine residue 397 was blocked by the FAK specific inhibitor PF 573228 in an ameloblast-like LS8 cell line. LS8 cells were treated with bRGDS PA after incubation with PF 573228 at a selected concentration ranging from 0, 0.05, 0.1, 0.3, 0.5, 0.9, 1.0, 1.5, 2.0 μM for 1 hr. Proteins from whole cell lysates were resolved and probed for phosphorylated FAK (p-FAK). FAK phosphorylation level revealed a sharp diminution at a FAK inhibitor concentration of approximately 300 nM;
(B) The effect of FAK inhibitor PF 573228 on the expression of mRNAs for amelogenin and C/EBPα in primary enamel organ epithelial (EOE) cells treated with different peptide amphiphile (PA) matrices using real-time PCR. Primary EOE cells were treated with either bRGDS PA or ScrRGDS PA for 4 hrs in the presence of 0.1% DMSO (vehicle control) or 500 nM FAK inhibitor PF 573228. The abundance of mRNAs for amelogenin and C/EBPα was markedly up regulated when cells were treated with bRGDS PA (bRGDS PA+DMSO), while the FAK inhibitor at 500 nM (bRGDS PA+FAK Inh) reduced the stimulating effect of bioactive PA on amelogenin (p=0.03, ANOVA), and C/EBPα expression (p=0.006, ANOVA), compared with mRNA abundance levels for the ScrRGDS PA treated group in either the presence or absence of PF 573228 (ScrRGDS PA+DMSO; ScrRGDS PA+FAK Inh).

(A) Confocal immunofluorescent analysis of primary enamel organ epithelial cells treated with ScrRGDS PA for 2 min using phospho-FAK Tyr397 (p-FAK) antibody (green), Alexa Fluor 594 phalloidin for F-actin (red) and DAPI for nuclei (blue). Slight stimulation of p-FAK was located mainly in the cytoplasm (arrowhead). Panel A1 is a higher magnification of the box outlined in panel A;
(B) Confocal immunofluorescent analysis of primary enamel organ epithelial cells, treated with bRGDS PA for 2 min using p-FAK antibody (green), Alexa Fluor 594 phalloidin for F-actin (red) and DAPI for nuclei (blue). Abundant stimulation of p-FAK, distributed in both the cytoplasm and cellular protrusions (arrowheads). Panel B1 is a higher magnification of the box outlined in panel B;
(C) Western blot analysis of protein from primary enamel organ epithelial cells to identify phospho-FAK Tyr397 (p-FAK) and total FAK. Cells were treated for 2- or 15-min with ScrRGDS PA or bRGDS PA or grown on tissue culture (TC) as a sham control. Equal amounts (15 μg) of lysate for each sample were used in each lane and probed with p-FAK or FAK antibodies. GAPDH is used as loading control;
(D) Densitometry tracing of phospho-FAK Tyr397 (p-FAK) (top panel) and total FAK (bottom panel) detected by Western blot analysis shown in panel D. The p-FAK and total FAK levels were normalized to GADPH values;
(E) The ratio of activated p-FAK to total FAK was significantly increased in enamel organ epithelial cells treated with bRGDS PA within 2 min (p<0.05, ANOVA) and returned to basal levels within 15 min.

(A) Peptide amphiphiles were injected into the enamel organ epithelia of an E18.5 mouse incisor grown on a nitrocellulose disc in organ culture. bRGDS PA matrix was mixed with a rhodamine containing PA (20:1) to permit fluorescent detection of the injection site (arrowhead) along the rostral-caudal gradient of incisor development and one that corresponds to an early secretory stage of enamel formation;
(B) A representative cross-sectional view of a tissue section from a bRGDS PA injected incisor stained with hematoxylin and eosin. A regenerated enamel nodule (arrowhead) was formed at the PA injection site after transplantation under the kidney capsule for an 8-week period. The cross sectional view of the nodule reveals a rosette of polarized dental epithelial cells (*) that surround a nodule of regenerated enamel matrix. rEn: regenerated enamel; Am: ameloblast; En: Enamel;
(C) Detection of amelogenin mRNA and ameloblast-specific transcription factor, C/EBPα in primary enamel organ epithelial (EOE) cells treated with different PAs by real-time PCR. Amelogenin transcripts were approximately 4 times as abundant in primary EOE cells grown on bRGDS PA compared with identical EOE cells grown on plastic (tissue culture plates, TC) or scrambled RGDS PA (ScrRGDS PA) (p < 0.05, ANOVA). C/EBPα transcripts were twice as abundant in primary EOE cells grown on bRGDS PA compared with cells grown on plastic (TC) or ScrRGDS PA (p < 0.01, ANOVA).

(A) Confocal immunofluorescent analysis of primary enamel organ epithelial (EOE) cells treated with ScrRGDS PA for 15 min, using a phosphorylated SAPK/JNK Thr183/Tyr185 (p-SAPK/JNK) antibody (green) and Alexa Fluor 594 phalloidin for F-actin (red);
(B), primary EOE cells treated with bRGDS PA for 15 min. Compared with primary EOE cells treated with control (scrambled) PA matrix, EOE cells treated with the integrin-binding bRGDS PA showed an increase in p-SAPK/JNK that accumulated in both the cytoplasm and nuclei (arrowhead);
(C), primary EOE cells were treated with bRGDS PA for 15 min in the presence of the JNK inhibitor SP 600125 at 0.9 μM, a concentration that demonstrates clear inhibition of the p-SAPK/JNK signal by Western blot analysis;
(D) Western blot analysis of p-SAPK/JNK in primary enamel organ epithelial (EOE) cells treated with control PA matrix (ScrRGDS PA) or with the bioactive bRGDS PA detected by antibody specific to p-SAPK/JNK Thr183/Tyr185. The intensity of the detected p-SAPK/JNK band was normalized to a constitutively expressed protein, GAPDH. The level of p-SAPK/JNK protein was approximately 3-times as abundant in primary EOE cells treated with bRGDS PA compared with ScrRGDS PA treated cells (*p<0.01, student t-test);
(E) The effect of JNK inhibitor SP 600125 on the protein levels for p-SAPK/JNK and phosphorylated c-Jun Ser63 (p-c-Jun) in primary enamel organ epithelial (EOE) cells treated with different PA matrices by Western blot analysis. Primary EOE cells were pre-incubated with 0.9 μM JNK inhibitor SP600125 for 1 hr, and then treated with bRGDS PA or ScrRGDS PA for 15 min. The expression of p-SAPK/JNK and p-c-Jun expression was markedly up regulated when cells were treated with bRGDS PA, while treatment with the JNK inhibitor SP600125 at 0.9 μM significantly reduced the activation of the bioactive bRGDS PA on p-SAPK/JNK and p-c-Jun.

(A) Graphical representation of the cis-elements within 5′-proximal region of the mouse amelogenin promoter. The software program “Biobase” was used to identify the “TGACT”-cis element sequence for c-Jun which is shown outlined by boxes located at c-Jun1 (-213nt) and c-Jun2 (-364nt) and the C/EBPα binding “GAAA” element is outlined by box located at around -70nt upstream of the transcription initiation site (TSS, right handed arrow) within the 5′-proximal region of the mouse amelogenin promoter;
(B) Ameloblast-like LS8 cells were transiently transfected with 250 ng of the full-length amelogenin promoter reporter construct, p2207-luc and with variable mass of the expression plasmid encoding c-Jun starting at 200 ng (lane 2), 500 ng (lane3), 1000 ng (lane4) or with 750 ng empty vector (pcDNA3, lane1) as the control. The transcriptional activity of the amelogenin promoter reporter construct p2207-luc activation was measured using a dual reported assay for Renilla luciferase (transfection control) or firefly luciferase from the amelogenin promoter and normalized to the Renilla value. The exogenous c-Jun activated the transcription activity of amelogenin promoter reporter in a dose dependent manner;
(C) Whole cell lysates from a concentration gradient for c-Jun construct transfected LS8 cells, resolved in size and detected with a specific c-Jun antibody. Increasing mass of the transfected c-Jun construct resulted in increased abundance of c-Jun protein in the LS8 cells;
(D) Schematic representation of site-directed mutagenesis generated in the 5′-proximal region of the mouse amelogenin promoter in the reporter construct. Nucleotide position -213 and -364 represent the c-Jun binding sites upstream of the transcription initiation site (TSS, right handed arrow) in the 5′-proximal region of the mouse amelogenin promoter, where the c-Jun binding sites were mutated as shown in bold font;
(E) Site-directed mutagenesis of c-Jun binding sites on the regulation of amelogenin transcription activity. Ameloblast-like LS8 cells were transiently transfected with 200 ng of Wild type (WT) or mutated (MUT) amelogenin promoter-luciferase reporter constructs in the presence of 500 ng of the expression plasmid encoding c-Jun. Reporter constructs with either the single mutation site at residue 213 (MUT (213)-luc) or at the residue 364(MUT (364)-luc) were generated. A reporter construct with double mutations MUT (213)-MUT (364)-luc was generated at the -213nt and -364nt c-Jun binding sites. Mutation at -213nt and/or -364nt upstream of the transcription initiation site of the mouse amelogenin promoter resulted in significant decrease in amelogenin transcription activity (*p<0.01; ** p<0.01; *** p<0.01, student t-test), indicating amelogenin mRNA expression is regulated by c-Jun;
(F) The endogenous amelogenin gene is transcriptionally activated by c-Jun. Amelogenin gene expression from the genomic locus was increased about 1.5 fold.