Expression And Regulation Of PTHrP And Its Receptor In Sika Deer Antler

The vast majority of mammalian organs are hard to regenerate after losing, deer antlers are theonly mammalian appendages to be lost and completely regenerated. Deer antler grows very rapidly,recent evidences suggest that several development pathways involved in antler regeneration, and atthe same time these pathways are also known to play a very important role in the bone regenerationand development of other lower vertebrates. However, to date, the exploring about the mechanismof antler regeneration and development is not very in-depth.Parathyroid hormone-related peptide (PTHrP) was originally identified as a factor involved inhumoral hypercalcemia of maligaancy. PTHrP and parathyroid hormone (PTH) share a highlyhomologous N-terminal region, and both peptides activate a common PTH/PTHrP receptor (PPR),which is G protein-coupled receptor. PTHrP regulate cell proliferation, differentiation andapoptosis by the autocrine or paracrine through the PPR. In growth plates, PTHrP plays a crucialrole to regulate chondrocytes differentiation. It could maintain chondrocytes in a proliferative stateand prevent premature chondrocytes hypertrophy. PTHrP-deficient mice showed acceleratedchondrocyte differentiation, while overexpression of PTHrP in chondrocytes could delaychondrocyte terminal differentiation.The aim of this study was to investigate the expression of PTHrP and its receptor in sika deerantler using in situ hybridization. The results of in situ hybridization showed: the mRNAexpression of PTHrP and its receptor in the several tissues of the growing tip of Sika deer antlercould be detected, indicating that PTHrP and its receptor play a crucial regulating effect in Sikadeer antler regeneration and rapid growth. PPR mRNA in the sika deer antler was very similar tothat of PTHrP and only had differences with PTHrP in part tissues. There was no detectable PTHrPmRNA signal in the epidermis, but the mRNA expression of PPR in the epidermis could bedetected at a moderate level, which was mainly expressed in the basal cells and keratin cells ofstratum spinosum. It was suggested that PTHrP might contribute to regulate anlter skin layersregeneration by the paracrine signaling systems through the PPR. In the dermis, both PTHrP andPPR mRNA were highly expressed in the dermal fibroblasts, indicating that PTHrP involved in maintaining the high rate of mitotic activity of the dermal fibroblasts to make sure to produce alarge number of fibroblasts. Both PTHrP and PPR mRNA were not found in hair follicles andsebaceous glands suggesting that other growth factors might contribute to regulate theirdevelopment. In the cartilage, a high level of PTHrP and PPR mRNA signal was also detected.ThePTHrP and PPR mRNA were mainly expressed in the proliferating chondrocytes, chondroblast andblood vessel-associated cells of the cartilage columns, showing that PTHrP might involved inregulating cells differentiation in the cartilage. At the same time, we used cell culture method invitro, adding PTHrP (1-34) and several blockers to block PTHrP signaling pathway to research theeffect of PTHrP on the antler chondrocytes differentiation and the changes of its downstream genemRNA expression level. The results of cell experiment showed that antler chondrocytes werecultured in new medium on day3, and the non-adherent cells were removed. Then we observed theantler chondrocytes under inverted microscope. The results indicated that the chondrocytes showedclone-like growth and the cells were triangle or polygon. The results of Trypan blue stainingshowed that the cell activity was above90%. The results of HE staining showed that partial cellswere spindle. The results of real time PCR showed that the expression level of Col II mRNA didnot visibly change after the chondrocytes were treated with PTHrP (1-34) for different periods.However, Col IX mRNA expression gradually declined after PTHrP treatment and reached a nadirat24h, the expression level of Col X mRNA after PTHrP treatment was very similar to that of ColIX, indicating that PTHrP might promote the antler chondrocytes proliferation and inhibit theirmature and differentiation. The second generation cells were pretreated with several kinaseinhibitors for2h, respectively before the addition of PTHrP (1-34). The changes of Bcl-2, CyclinD1and Runx2mRNA expression levels were measured by real time PCR. The results showed thatthe expression level of PTHrP-induced Bcl-2was gradually enhanced and reached a peak at12h.However, the PTHrP stimulation of Bcl-2expression was dramatically altered by adding PKAinhibitor H-89, p38MAPK inhibitor SB20385and MEK inhibitor U0126. In other experimentalgroups, the results showed that while PTHrP alone stimulated the Bcl-2expression compared tountreated controls, the addition of JNK inhibitor SP600125only slightly downregulated thePTHrP-induced stimulation of Bcl-2expression, whereas PKC inhibitor GF109203X had nearly noeffect; after antler chondrocytes were treated with PTHrP, the expression level of Cyclin D1mRNA also gradually increased and reached a peak at12h. The PTHrP stimulation of cyclin D1gene expression was also significantly altered by addition of the H-89and GF109203X. In other experimental groups, the results showed that while PTHrP alone stimulated the cyclin D1geneexpression compared to untreated controls, the SB20385and SP600125only slightly altered thePTHrP-induced stimulation of cyclin D1gene expression, whereas U0126had nearly no effect;after antler chondrocytes were treated with PTHrP alone, the expression level of Runx2mRNAgradually decreased and reached a nadir at24h. The inhibitory effect of PTHrP on Runx2wasrescued effectively by the addition of H-89, SB203850, U0126or PI3K inhibitor LY294002respectively, while PKC had no effect. These results suggest that PTHrP regulation of Bcl-2ismainly though PKA, p38MAPK and MEK signaling pathways in antler chondrocytes; PTHrPregulation of cyclin D1is mainly PKA and PKC dependent in antler chondrocytes; PTHrPinhibition of Runx2is mainly through PKA, p38MAPK, MEK and PI3K signaling pathways inantler chondrocytes.The present study will provide important theoretical foundation to further understand thefunction of PTHrP in antler regeneration and the regulation of rapid growth.