ABSTRACT
Objective:To explore the mechanism of highly expressed primary cilia in tibial growth plate chondrocytes accelerating chondrocytes differentiation in young rats with chronic renal failure (CRF).Methods:Forty male 4-week-old SD rats weighing (98±3) g were randomly divided into control group (intragastric administration with distilled water, n=20) and CRF group (intragastric administration with adenine suspension 150 mg·kg -1·d -1, n=20). All the young rats were sacrificed after continuous gavage for 6 weeks. The length of the growth plate was measured with histological sections. Immunofluorescence (IF) was used to detect the expression rate of primary cilia and the level of β-catenin, the key protein of Wnt/β-catenin signaling pathway in tibial growth plate chondrocytes. Chondrocytes isolated from growth plate in two groups were cultured in vitro to P3 generation, and the expression rate of primary cilia in chondrocytes, the levels of Indian hedgehog (IHH) and glycogen synthase kinase 3β (GSK3β) were detected by IF. Co-immunoprecipitation was used to detect the relationship between IHH and GSK3β. Results:Compared with the control group, the relative length of the growth plate was shorter in histological sections [(0.51±0.11) vs (1.00±0.08), t=16.11, P<0.001], the expression rate of primary cilia was higher [(26.3±5.5)% vs (7.6±1.9)%, t=14.37, P<0.001], and the level of β-catenin increased [(7.1±2.0) scores vs (3.6±1.0) scores, t=7.10, P<0.001] in CRF group. In vitro, the expression rate of primary cilia was higher in CRF group chondrocytes [(31.4±8.2)% vs (12.5±3.1)%, t=9.64, P<0.001] than that in control group. The level of IHH in CRF group increased than that in control group [(1 360±270) vs (310±84), t=16.61, P<0.001]. There was no significant difference in GSK3β level of chondrocytes between the two groups [(850±195) vs (780±140), t=1.30, P=0.200]. There was a direct interaction between IHH and GSK3β in CRF group chondrocytes. Conclusions:The expression levels of primary cilia and related protein IHH increase in tibial growth plate chondrocytes of CRF young rats. The IHH protein plays a direct interaction with GSK3β protein, Wnt/β-catenin signaling pathway antagonist, which leads to the activation of Wnt/β-catenin signaling pathway and final accelerated differentiation of chondrocytes. The rapid differentiation of chondrocytes causes the closing trend of growth plate.
ABSTRACT
<p><b>OBJECTIVE</b>To determine the influence of altered masticatory loading on Indian hedgehog (Ihh)-parathyroid hormone-like related protein (PThrP) pathway in the condylar cartilage of growing rabbits.</p><p><b>METHODS</b>A total of 48 10-day-old rabbits were randomly divided into two groups and fed different kinds of food, such as solid diet and soft diet. The animals were sacrificed after 2, 4, 6, and 8 weeks. Difference of Ihh and PThrP expression levels induced by altered masticatory loading was tested by hematoxylin-eosin (HE), immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR).</p><p><b>RESULTS</b>The thickness of condylar cartilage and expression levels of Ihh and PThrP proteins and mRNA of the solid diet groups exceeded those of the soft diet groups. The decreasing tendencies of the expression levels of Ihh and PThrP proteins and mRNA were observed at 2, 4, 6, 8 weeks.</p><p><b>CONCLUSIONS</b>Low masticatory loading may delay or inhibit the development of condylar cartilage and its growing factors Ihh and PThrP. Therefore, masticatory loading plays an important role in the development of condylar cartilage.</p>
Subject(s)
Animals , Rabbits , Blotting, Western , Cartilage , Chondrocytes , Hedgehog Proteins , Immunohistochemistry , Mastication , Parathyroid Hormone , Real-Time Polymerase Chain ReactionABSTRACT
The roles of Indian hedgehog (Ihh) signaling pathway in the proliferation and apoptosis of precartilaginous stem cells (PSCs) were investigated.PSCs,labeled with fibroblast growth factor receptor 3 (FGFR-3),were isolated from neonatal rats by immanomagnetic separation.After identifi-cation with FGFR-3 and Col Ⅱ,the cells were incubated with different concentrations of cyclopamine (cyclo),the specific inhibitor of lhh signaling pathway.The morphologic changes of the cells were observed under the inverted phase contrast microscope.The mRNA expression levels of Ibh,para-thyroid hormonerelated peptide (PTHrP),protein Patched (Ptch),Bcl-2 and p21 were detected by RT-PCR.The protein expression levels of Ihh and Ptch were measured by Western blot.MTT assay was used to examine the effects of cyclo on proliferation of PSCs.Apoptosis rate of PSCs was exam-ined by Annexin V/PI assay of flow cytometric analyses.After PSCs were incubated with cyclo,ob-vious morphologic changes were observed as compared with the control group.The mRNA expres-sion levels of PTHrP,Ptch and Bcl-2 were decreased to varying degrees in a cyclo dose-dependent manner.However,the expression levels of lhh and p21 mRNA were increased.The protein expres-sion of Ptch and Ihh had the same change as the mRNA expression.Meanwhile,cyclo could obvi-ously inhibit the proliferation and promote the apoptosis of PSCs.The results indicated that Ihh sig-naling pathway plays an important role in regulating the proliferation and apoptosis of PSCs,which is probably mediated by Bcl-2 and p21.
ABSTRACT
The mandibular condyle formation during temporomandibular joint (TMJ) development exhibits endochondral bone formation, and the elongation process is dependent on the normal cartilage proliferation and differentiation. Retinoids are important for maturation of growth-plate chondrocytes, but the identity of their downstream effectors remains unclear. In this study, we carried out a series of studies at the cellular, biochemical, and molecular levels to determine whether, and if so how, retinoid signaling is related to the expression and function of Indian hedgehog (Ihh) in chondrocyte proliferation. First we analyzed the RA receptor (RAR) and Ihh expression pattern in E18 mandibular condyle. <i>RARα<i/> and RARβ</i> mRNA were characterized in the perichondrium around the condyle, whereas <i>RARγ</i> mRNA was expressed in the immature and prehypertrophic chondrocytes and the expression was overlapped with Ihh gene expression. Next we established a high-density culture model of chick cephalic chondrocytes in the prehypertrophic stage. We found that all-trans retinoic acid (RA) induced Ihh mRNA gene expression in this system. The RA pan-antagonist Ro 41-5253 inhibited both endogenous and RA-induced Ihh mRNA in a dose-dependent manner. The Ihh mRNA expression induced by RA required <i>de novo</i> protein synthesis, and was mediated by RARγ. Immunoblots showed that the prehypertrophic chondrocytes contained sizable levels of phosphorylated p38 mitogen-activated protein (MAP) kinase that were time- and dose-dependently increased by the RA treatment. Experimental p38 inhibition led to a severe drop in baseline and RA-stimulated Ihh expression. Exogenous recombinant Ihh stimulated the proliferation of proliferating chondrocytes, whereas RA inhibited the proliferation of these chondrocytes through p38 MAPK. Retinoids appear to play a primary role in controlling both the expression and function of Ihh in prehypertrophic chondrocytes and do so via p38 MAP kinase.