Substance P (SP) is known to be expressed in the nerve fibers of dental pulp and periodontal tissues. It was recently reported that SP expression increased in response to orthodontic force. In the present study, we investigated the effect of SP on expression of mineralization markers and heme oxygenase-1 (HO-1) in human immortalized periodontal ligament (IPDL) cells. Cell viability was measured using a 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of mineralization markers, including alkaline phosphatase (ALP), osteonectin (ON) and bone sialoprotein (BSP), and heme oxygenase-1 (HO-1) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. SP did not significantly change human IPDL cell viability, with the exception of the 24 hour treatment group. Treatment of human IPDL cells with 10-10 to 10-⁴M SP upregulated mineralization marker and HO-1 expression in a time- and concentration-dependent manner. Our results suggest that SP may modulate osteoblastic cell differentiation of human IPDL cells through a mechanism involving HO-1 expression.

Previous in vi tro studies demonstrated that H202 or carbamide peroxide cou ld penetrate i nto pul p chambers through enamel and dentin (Benetti et a l., 2004; G okay et a l. , 2004‘ Suli eman et al .. 2005) ‘ Recently. Lee et al.(2006) demonstrated that H20Z enhanced the diffe rentiation of odontoblast like cell line, whereas it inhibited osteogenic diffe rentiation in pre 。steobl astic cell line, as seen by its efl"ecLs on an early difï"erentiation marker. ALP activity. I-lowever. the effects of HZ02 have not been well elucidated in primary cultured human pulp cells ln th is study‘ we investigated whether HO- 1 is involved in H20 2-induced cytotoxicity and examined the production 0 1" dent in sia lophosphoprotein (DSPP) and other minera li zation markers, in human pulp cells H20Z dec1'eased cell viabili ty. but increased HO-l and DSPP expression in a concentra t ion and time dependent manner. Inhibitors of guanylate cyclase, PI3K. ERK, and p38 MAP kinase blocked J-!?,0 2- induced cytot oxicity and the expression of HO-1 and DSPP mRNAs in pulp cells. These data suggest that t he induction of HO-l by H202 in pu lp cells plays a protective role against the cytotoxic effects of H202 and stimulates DSPP expression. resulting in prematu re oclontoblast differentiation th rough pathways t hat involve cGMP. p38. ancl ERK

Although substance P (SP). a potent pro-inflammatory peptide, is involved in inflammation and immune responses, the effect of SP 011 the expression of macl'ophage inJlammatol'Y protein 3a (MIP-3a. CCL20) in periodontal ligament (PDL) cells a l'e unknown Equally as enigmatic is the link between SP. the stress protein heme oxygenase-l (HO-l) , and CCL20 product ion. We investigated whether SP induces the release of chemokine CCL20 from irrunortalized POL (IPDL) cells. and further claif’y SP mediated pathways . We also exarnined the relationship between HO-l and CCL20 by treating POL cells with SP Incubating IPOL cells with SP incl'eased ex pl'ession of CCL20 mRNA and CCL20 protein in a dose-time dependent manner. Highly selective p38 and ERKl/2 inhibitors abl'ogated SP-induced expression of CCL20 lD IPOL cells SP is also responsible fo l' ini tiating phosphorylation of I/( B‘ degl'adation of IK B. and activation of NF-/( B. SP induced expression of HO-l in both a concentration- and time-dependent manner. and CCL20 refl ected similal' patterns. The inductive effects of SP on HO-l and CCL20 were enhanced by HO- l inducer hemin and the membrane-permea ble cGMP analog 8-bromo-cGMP Conversely, this pathway was inhi bited by the HO-l inhibitor zinc Pl'otoporphyrin IX (ZnPP IX) and the selective inhibitor of guanylate cyclase‘ 1H- [1. 2. 4]uxad iazole[4, 3-alquinoxal i n- 1-one (ODQ) We report hel'ein the pathway that connects SP a long with other modulators 0 1' neuroimmunoregulationto the induction of HO-1 and the inflanunatol'y mediatol' MIP- 3a /CCL20 in IPDL cel ls. which play an impol'tant role in the development 0 1' pe- I'iodontitis or inflammation during ol'thodontic tooth movement