DPP and DSP are Necessary for Maintaining TGF-β1 Activity in Dentin

Abstract

Porcine dentin sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentin. It is processed by proteases into 3 independent proteins: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We fractionated DPP and DSP along with TGF-β activity by ion exchange (IE) chromatography from developing pig molars and measured their alkaline phosphatase (ALP)-stimulating activity in human periodontal (HPDL) cells with or without TGF-β receptor inhibitor. We then purified TGF-β-unbound or -bound DPP and DSP by reverse-phase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-β isoform bound to DPP and DSP was identified as being TGF-β1 by both ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-β1 (CF-hTGF-β1) with TGF-β-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-β1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-β1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-β to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-β1. We conclude that both DPP and DSP help retain TGF-β1 activity in porcine dentin.

Keywords: HPLC; cell culture; extracellular matrix (ECM); isolation and purification; phosphophoryn; tooth.

Figure 1.

Isolation of DSPP-derived proteins in porcine molar dentin. (A) Q-Sepharose chromatograms showing absorbance at 230 nm for AN extracts from dentin of porcine developing molars (150 mg). Downward-pointing arrows are the starting point of the step gradient with 0.2, 0.5, and 2 M NaCl. (B) SDS-PAGE (4% to 20% gradient gel) stained with Stains-All showing each tube on a Q-Sepharose chromatogram. (C) Western blots showing each tube on a Q-Sepharose chromatogram with specific antibodies against N-terminal dentin sialoprotein (N-DSP Ab, left) and dentin glycoprotein (DGP Ab, right), showing isolated DSP in Q3 and Q4 fractions. (D) ALP-inducing activity of HPDL cells exposed by Q3 and Q4 fractions and TGF-β1 (0.3 ng/mL) without (-) or with (+) SB431542. Data are means ± SE of 3 culture wells.

Figure 2.

Isolation of TGF-β1-unbound and -bound DPP and DSP in porcine molar dentin. (A) RP-HPLC chromatograms showing absorbance at 220 nm for ANQ3 and ANQ4 (5 mg each) fractionated by IE chromatography and for CF-hTGF-β1 (1 µg). (B) ALP-inducing activity of HPDL cells exposed by fractions 8-23 in ANQ3, ANQ4, and CF-hTGF-β1. TGF-β1 (0.3 ng/mL) is used as a positive control. Data are means ± SE of 3 culture wells. (C) SDS-PAGE (4% to 12% gradient gel) stained with Stains-All showing each tube in ANQ3 fractionated by RP-HPLC. (D) SDS-PAGE (4% to 12% gradient gel) stained with Simply Blue (top) and Western blots (bottom) used specific antibodies against N-terminal dentin sialoprotein, showing each tube in ANQ4 fractionated by RP-HPLC. (E) ELISA for the detection of TGF-β1 in combined fractions 17-20 in ANQ3 and ANQ4 enhanced ALP-inducing activity in HPDL cells. This figure is available in color online at http://jdr.sagepub.com.

Figure 3.

In vitro binding study of CF-hTGF-β1 for DSP, DPP, and type I collagen. (A, C, F, I) IE-HPLC chromatograms showing absorbance at 280 nm for (A) CF-hTGF-β1 only, (C) TGF-β1-unbound-DSP only (DSP only, top) and TGF-β1-bound DSP after binding experiment (DSP + CF-hTGF-β1, bottom), (F) TGF-β1-unbound-DPP only (top) and TGF-β1-bound DPP after binding experiment (DPP + CF-hTGF-β1, bottom), and (I) type I collagen only (Col I only, top) and TGF-β1-bound collagen after binding experiment (Col I + CF-hTGF-β1, bottom). (B, D, G, J) ALP-inducing activity of HPDL cells exposed by (B) fractions 4 to 14 in CF-hTGF-β1 only, (D) TGF-β1-unbound-DSP only (DSP only, top) and TGF-β1-bound DSP (DSP + CF-hTGF-β1, bottom), (G) TGF-β1-unbound-DPP only (DPP only, top) and TGF-β1-bound DPP (DPP + CF-hTGF-β1), and (J) fractions 4 to 13 in type I collagen only (Col I only, top) and TGF-β1-bound collagen (Col I + CF-hTGF-β1, bottom). The recombinant human TGF-β1 (TGF-β1) (0.3 ng/mL) was used as positive control. Data are means ± SE of 3 culture wells. (E, H, K) SDS-PAGE (4% to 12% gradient gel) stained with Stains-All showing (E) DSP in fractions 4 to 14, (H) DPP in fractions 4 to 14, and (K) stained with Simply Blue showing type I collagen in fractions 4 to 13 isolated by IE-HPLC after binding to CF-hTGF-β1. This figure is available in color online at http://jdr.sagepub.com.