Table 1.
Growth of rats treated with vehicle (C), rapamycin (RAPA) or with rapamycin and growth hormone (RGH).
Figure 1.
GH effects on longitudinal growth rate and growth plate morphology.
(A) Representative images showing the distance between the metaphyseal end of the growth cartilage and the fluorescent calcein front, which indicates longitudinal bone growth rate during the last 3 days of the study, in control rats (C), rats treated with rapamycin (RAPA) or rapamycin and GH (RGH). (B) Representative alcian blue/safranine stained sections showing tibial growth cartilage morphology of C, RAPA and RGH animals. (C) Representative Von Kossa stained sections showing the pattern of extracellular matrix mineralization in proximal tibial growth plate of C, RAPA and RGH animals. Mineralized transverse septa (Red arrows) were often found in RAPA and RGH animals and not in C group. Magnification bars = 100 µm.
Table 2.
Growth plate characteristics of rats treated with vehicle (C), rapamycin (RAPA) or with rapamycin and growth hormone (RGH).
Figure 2.
GH effects on growth plate cell proliferation and angiogenesis.
(A) Representative images of BrdU immunodetection in the proliferative zone of the epiphyseal cartilage of control rats (C), rats treated with rapamycin (RAPA) or rapamycin and GH (RGH). (B) Representative sections of proximal tibial growth plates stained with picrosirius red/alcian blue showing trabeculae and vascular sprouts arrangement in the primary spongiosa of C, RAPA and RGH animals. Transverse unresorbed septa are indicated with yellow arrows. Vascular sprouts are indicated with red arrows. (C) Representative sections of proximal tibial growth plates stained with tartrate-resistant acid phosphatase (TRAP) showing positive chondroclasts/osteoclasts in the chondro-osseous junction of C, RAPA and RGH animals. Representative images of immunohistochemistry (D) and in situ hybridization (E) experiments showing VEGF expression in growth plates of C, RAPA and RGH animals. Magnification bars = 100 µm.
Figure 3.
GH effects on chondrocyte autophagy.
(A) Immunofluorescent detection of LC3, a marker of autophagy, in the growth plates of rats treated with rapamycin (RAPA) or rapamycin and GH (RGH). Fluorescent signal was observed in prehypertrophic chondrocytes often displaying a punctuate distribution (white arrows in B). (C) Western blot of LC3-I (18 kDa) and LC3-II (16 kDa) in the growth cartilage of rats treated with rapamycin (RAPA) or rapamycin and GH (RGH). GAPDH was used as loading control. Image is representative of three blots giving similar results.
Figure 4.
GH effects on chondrocyte metabolism.
(A) Periodic acid-Schiff (PAS) reaction in the proximal tibial growth plate of control rats (C), rats treated with rapamycin (RAPA) or rapamycin and GH (RGH). Magnification bars = 100 µm. (B) Western blot of p-GSK3β (Ser9) and GSK3β in the growth cartilage of C, RAPA and RGH animals. (C) Western blot of β-catenin in the growth cartilage of C, RAPA and RGH animals. (D) Western blot of p-AKT (Thr308), AKT, p-ERK1/2 (Thr202/Tyr204) and ERK1/2 in the growth cartilage of C, RAPA and RGH animals. GAPDH was used as loading control. Images are representative of three blots giving similar results. (E) Densitometry analysis of the western blot experiments shown in B,C and D. At least four animals per group were used per experiment and each assay was repeated at least three times. aMeans statistically different from C group (P≤0.05). bMeans statistically different from RAPA group (P≤0.05).
Figure 5.
Proposed mechanism of the stimulating effect of GH on the hypertrophy of growth cartilage chondrocytes.
GH would signal through PIP3/Akt and MAPK pathways to phosphorylate and inactivate GSK3β in growth plate chondrocytes. This would lead to an increase of glycogen synthesis and stabilization of β-catenin that, eventually, would enhance chondrocyte hypertrophy.