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, 189 (1), 132-146

Novel mTORC1 Mechanism Suggests Therapeutic Targets for COMPopathies

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Novel mTORC1 Mechanism Suggests Therapeutic Targets for COMPopathies

Karen L Posey et al. Am J Pathol.

Abstract

Cartilage oligomeric matrix protein (COMP) is a large, multifunctional extracellular protein that, when mutated, is retained in the rough endoplasmic reticulum (ER). This retention elicits ER stress, inflammation, and oxidative stress, resulting in dysfunction and death of growth plate chondrocytes. While identifying the cellular pathologic mechanisms underlying the murine mutant (MT)-COMP model of pseudoachondroplasia, increased midline-1 (MID1) expression and mammalian target of rapamycin complex 1 (mTORC1) signaling was found. This novel role for MID1/mTORC1 signaling was investigated since treatments shown to repress the pathology also reduced Mid1/mTORC1. Although ER stress-inducing drugs or tumor necrosis factor α (TNFα) in rat chondrosarcoma cells increased Mid1, oxidative stress did not, establishing that ER stress- or TNFα-driven inflammation alone is sufficient to elevate MID1 expression. Since MID1 ubiquitinates protein phosphatase 2A (PP2A), a negative regulator of mTORC1, PP2A was evaluated in MT-COMP growth plate chondrocytes. PP2A was decreased, indicating de-repression of mTORC1 signaling. Rapamycin treatment in MT-COMP mice reduced mTORC1 signaling and intracellular retention of COMP, and increased proliferation, but did not change inflammatory markers IL-16 and eosinophil peroxidase. Lastly, mRNA from tuberous sclerosis-1/2-null mice brain tissue exhibiting ER stress had increased Mid1 expression, confirming the relationship between ER stress and MID1/mTORC1 signaling. These findings suggest a mechanistic link between ER stress and MID1/mTORC1 signaling that has implications extending to other conditions involving ER stress.

Figures

Figure 1
Figure 1
Midline 1 (MID1) is up-regulated in mutant cartilage oligomeric matrix protein (MT-COMP) mouse growth plate chondrocytes. Mid1 mRNA level was assessed by microarray and Mid1 protein by immunohistochemistry from P1 to P28. A:Mid1 expression in C57BL/6 mice (control; dark gray bars) was set to 1 and compared with the level of Mid1 expression in the MT-COMP mice (light gray bars). Growth plate chondrocytes from P1 to P14 show more Mid1 mRNA in the MT-COMP chondrocytes compared to control. B–I: Immunostaining of control C57BL/6 and MT-COMP growth plates with Mid1 (red) and DAPI (blue) stained nuclei from P1 to P28. Arrowheads mark the cells that are enlarged in the insets. Data are expressed as means ± SD. n = 3 mice per group (A). P < 0.05. Original magnification: ×400 (BI); ×800 (insets).
Figure 2
Figure 2
Midline (MID)-1 is increased in human pseudoachondroplasia (PSACH) chondrocytes and by endoplasmic reticulum stress in rat chondrosarcoma (RCS) cells. A–L: Chondrocytes from control and three different PSACH patients [with D511Y, G427E, and D469del cartilage oligomeric matrix protein (COMP) gene mutations] were grown in a three-dimensional culture system as previously described, to generate nodules that were immunostained with MID1 antibody. DAPI (blue signal) marks the nuclei of chondrocytes. Arrowheads indicate the cells that are enlarged in the insets; dashed circles indicate the nuclei in the insets. M:Mid1 mRNA levels in RCS cells treated with thapsigargin or tunicamycin (gray bars) are compared to that in untreated cells (control; black bars). Data are expressed as means ± SD. n > 100 cells (AL); n = 9 (M). ∗∗P < 0.005, ∗∗∗P < 0.0005. Original magnification: ×600 (AL); ×1500 (insets).
Figure 3
Figure 3
Tumor necrosis factor (TNF)-α and TNF-related apoptosis-inducing ligand (TRAIL) are increased in mutant cartilage oligomeric matrix protein (MT-COMP) mouse growth plate chondrocytes. A–J: TNFα and TRAIL proteins were evaluated by immunohistochemistry in growth plates from mice at P28. K: Mid1 and Trail mRNA levels in rat chondrosarcoma (RCS) cells were assessed by quantitative real-time RT-PCR treated with TNFα for either 24 or 72 hours. Mid1 and Trail expression in RCS cells (gray bars) was set to 1 and compared with RCS cells treated with 200 ng/mL TNFα for either 24 or 72 hours (black bars). Data are expressed as means ± SD. n = 9 for all groups. P < 0.05. Scale bars = 100 μm.
Figure 4
Figure 4
Increased midline 1 (MID1) decreases protein phosphatase (PP)-2a in mutant cartilage oligomeric matrix protein (MT-COMP) mouse growth plate chondrocytes. A–Y: MID1 (A–E), PP2A (F–J), phosphorylated AKT (K–O), phosphorylated mammalian target of rapamycin (pmTOR; P–T), and phosphorylated S6 ribosomal protein (pS6; U–Y) assessed by immunohistochemistry in P28 control (C57BL/6), MT-COMP with no treatment, MT-COMP treated with aspirin or resveratrol, and MT-COMP/CHOP-/- growth plate chondrocytes. AKT, mTOR, and pS6 are all components of the mTORC1 signaling pathway. Z: RT-PCR performed on RNA extracted from tuberous sclerosis complex (TSC)-1–null, TSC2-null, TSC1/2–double null, and control mouse brains. Data are expressed as means ± SD. n > 3 (AY); n = 3 (Z). P < 0.05, ∗∗∗P < 0.0005 versus control. Scale bar = 100 μm (all images).
Figure 5
Figure 5
Rapamycin treatment of mutant cartilage oligomeric matrix protein (MT-COMP) mice decreases intracellular retention of COMP and mammalian target of rapamycin complex 1 (mTORC1) signaling but not inflammation. Control, untreated MT-COMP, and rapamycin-treated MT-COMP growth plates at P28 immunostained with antibodies for phosphorylated S6 ribosomal protein (pS6) (A–C), human (h) COMP (D–F), IL-16 (G–I), and DNA proliferation cell nuclear antigen (PCNA) (J–L). Rapamycin treatment reduces pS6 and intracellular COMP and improves chondrocyte proliferation. The inflammatory marker IL-16 is not reduced by rapamycin treatment. Scale bars = 100 μm.
Figure 6
Figure 6
Glioma-associated oncogene homolog 2 (Gli2) and parathyroid hormone-related protein (PTHrP) are increased with mammalian target of rapamycin complex 1 (mTORC1) signaling in mutant cartilage oligomeric matrix protein (MT-COMP) growth plates. Control (C57BL/6), untreated MT-COMP, MT-COMP treated with resveratrol or aspirin, and MT-Comp/CHOP−/− growth plates from P28 mice were immunostained for phosphorylated GLI2 (A–E) and PTHrP (F–J). Scale bar = 100 μm (all images). CHOP, CCAAT/enhancer-binding proteinehomologous protein.
Figure 7
Figure 7
Overexpression or knockdown of Mid1 alters Chop mRNA levels. A and B: Rat chondrosarcoma (RCS) cells that express human mutant cartilage oligomeric matrix protein (h-MT-COMP) were transfected with midline (MID)-1 expression plasmid and Mid1 (A) and Chop (B) mRNA levels were assessed. C:Mid1 siRNAs were transfected into RCS cells that express h-MT-COMP, resulting in decreased Mid1 levels. DH: mRNA levels of h-MT-COMP (D), Chop (E), Gadd34 (F), Gadd45a (G), and Ero1b (H) are shown. Data are expressed as means ± SD. n = 9 in each group. P < 0.05, ∗∗P < 0.005, and ∗∗∗P < 0.0005 versus MT-COMP.
Figure 8
Figure 8
Model depicting the roles of midline 1 (MID1), mammalian target of rapamycin, complex 1 (mTORC1), and parathyroid hormone-related protein (PTHrP) in mutant cartilage oligomeric matrix protein (MT-COMP) chondrocyte pathology. A: MT-COMP expression elicits endoplasmic reticulum (ER) stress through PERK/CHOP, which leads to oxidative stress and inflammation. The inflammatory process driven in part by tumor necrosis factor (TNF)-α increases TNF-related apoptosis-inducing ligand (TRAIL) and MID1. CHOP down-regulates general protein synthesis as part of the unfolded protein response and up-regulates AKT. AKT and MID1 stimulate mTORC1 signaling, along with a decrease in the mTORC1 brake, protein phosphatase (PP)-2A. mTORC1 signaling drives protein synthesis through protein S6 kinase/phospho-S6 and likely generates additional ER stress. mTORC1 up-regulates glioma-associated oncogene homolog 2 (zinc finger protein) (GLI)-2 and PTHrP, which alters chondrocyte proliferation and hypertrophy. B: Resveratrol counteracts several processes involved in the MT-COMP chondrocyte pathology including oxidative stress, inflammation, and MID1/α4 complex. Aspirin, a cyclooxygenase-2 inhibitor, dampens inflammation and diminishes the negative effects of MT-COMP expression (red). Rapamycin decreases intracellular retention of MT-COMP and mTORC1 signaling but does not improve inflammation and proliferation. Other drugs to potentially reduce the MT-COMP chondrocyte pathology are shown in blue. BHA, butylated hydroxyanisole; BIX, immunoglobulin heavy-chain–binding protein inducer X; CHOP, CCAAT/enhancer-binding protein–homologous protein; PBA, 4-phenylbutyrate; PDPK-1, protein 3-phosphoinositide-dependent protein kinase-1; PERK, protein kinase RNA-activated–like endoplasmic reticulum kinase TUDCA, tauroursodeoxycholic acid.
Supplemental Figure S1
Supplemental Figure S1
The endoplasmic reticulum (ER) stress drugs thapsigargin and tunicamycin increase midline 1 (MID1) in rat chondrosarcoma (RCS) cells. Mid1 and Chop mRNA levels in RCS cells treated with thapsigargin (A and B), tunicamycin (C and D), or peroxynitrite (E and F) were compared to that in untreated cells (control). CCAAT/enhancer-binding protein–homologous protein (CHOP) is an ER stress marker. Thapsigargin and tunicamycin increase Mid1 in a dose-dependent manner, and each experiment was repeated three times. Data are expressed as means ± SD. P < 0.05, ∗∗P < 0.005, and ∗∗∗P < 0.0005. versus control

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