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Modulation of TGFbeta 2 levels by lamin A in U2-OS osteoblast-like cells: understanding the osteolytic process triggered by altered lamins.

Evangelisti C, Bernasconi P, Cavalcante P, Cappelletti C, D'Apice MR, Sbraccia P, Novelli G, Prencipe S, Lemma S, Baldini N, Avnet S, Squarzoni S, Martelli AM, Lattanzi G - Oncotarget (2015)

Bottom Line: Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2.TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment.Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

View Article: PubMed Central - PubMed

Affiliation: Rizzoli Orthopedic Institute, Laboratory of Musculoskeletal Cell Biology, CNR Institute for Molecular Genetics, Unit of Bologna, Bologna, Italy.

ABSTRACT
Transforming growth factor beta (TGFbeta) plays an essential role in bone homeostasis and deregulation of TGFbeta occurs in bone pathologies. Patients affected by Mandibuloacral Dysplasia (MADA), a progeroid disease linked to LMNA mutations, suffer from an osteolytic process. Our previous work showed that MADA osteoblasts secrete excess amount of TGFbeta 2, which in turn elicits differentiation of human blood precursors into osteoclasts. Here, we sought to determine how altered lamin A affects TGFbeta signaling. Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2. TGFbeta 2 in turn, triggers the Akt/mTOR pathway and upregulates osteoprotegerin and cathepsin K. TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment. Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

No MeSH data available.


Related in: MedlinePlus

The Akt/mTOR pathway is modulated by lamins through TGFbeta 2(A) Western blot analysis of U2-OS cells transfected with mock or WT, R527H or L647 LMNA plasmids showing the amount of active Akt (Thr308p-Akt and Ser473p-Akt), p70S6K (p-p70S6K) and S6RP (p-S6RP) and their total amount (Akt, p70S6K and S6RP, respectively). Actin bands show equal loading. The corresponding densitometric analysis of immunoblotted bands is reported in (B) as mean values of three different experiments +/– standard deviation. (C) Neutralization of TGFbeta 2 activity in R527H U2-OS cells blocks the activation of the Akt/mTOR pathway. U2-OS expressing WT or R527H LMNA were subjected to western blot analysis. R527H U2-OS were left untreated (R527H) or treated with anti-TGFbeta 2 neutralizing antibody (right lane, TGFbeta2 Ab R527H) as detailed in the methods section. The corresponding densitometric analysis of immunoblotted bands is reported in (D) as mean values of three different experiments +/– standard deviation. (E) Phosphorylated ERK 1/2 (p-ERK 1/2) and ERK 1/2 levels in U2-OS 24 hours after transfection of mock (CTR), WT, R527H or L647R LMNA plasmids. ERK1/2 activity is reduced by expression of WT LMNA, but not by R527 LMNA. Statistically significant differences (P < 0.05) are indicated by asterisks in (in B, D, (F)). (G) Schematic representation of the TGFbeta 2-dependent signaling pathway affected by R527H-mutated LMNA expression and possible targets of drug intervention. R527H-mutated LMNA fails to regulate TGFbeta 2 levels causing elevated levels of TGFbeta 2 in U2-OS cells. Downstream events are activation of the Akt pathway and ERK 1/2 activation, which influence mTORC1 activity. The Akt/mTOR pathway can be blocked at diverse levels using MK2206 to inhibit Akt, RAD001 or statins (mevinolin) to inhibit mTORC1. Mevinolin also acts on mutated prelamin A avoiding protein farnesylation and favoring phosphorylation by Akt and lysosomal degradation of prelamin A (4, 5). RAD001 is also expected to trigger prelamin A degradation (33). Anti-TGFbeta 2 neutralizing antibody can be used to block the whole signaling pathway.
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Figure 3: The Akt/mTOR pathway is modulated by lamins through TGFbeta 2(A) Western blot analysis of U2-OS cells transfected with mock or WT, R527H or L647 LMNA plasmids showing the amount of active Akt (Thr308p-Akt and Ser473p-Akt), p70S6K (p-p70S6K) and S6RP (p-S6RP) and their total amount (Akt, p70S6K and S6RP, respectively). Actin bands show equal loading. The corresponding densitometric analysis of immunoblotted bands is reported in (B) as mean values of three different experiments +/– standard deviation. (C) Neutralization of TGFbeta 2 activity in R527H U2-OS cells blocks the activation of the Akt/mTOR pathway. U2-OS expressing WT or R527H LMNA were subjected to western blot analysis. R527H U2-OS were left untreated (R527H) or treated with anti-TGFbeta 2 neutralizing antibody (right lane, TGFbeta2 Ab R527H) as detailed in the methods section. The corresponding densitometric analysis of immunoblotted bands is reported in (D) as mean values of three different experiments +/– standard deviation. (E) Phosphorylated ERK 1/2 (p-ERK 1/2) and ERK 1/2 levels in U2-OS 24 hours after transfection of mock (CTR), WT, R527H or L647R LMNA plasmids. ERK1/2 activity is reduced by expression of WT LMNA, but not by R527 LMNA. Statistically significant differences (P < 0.05) are indicated by asterisks in (in B, D, (F)). (G) Schematic representation of the TGFbeta 2-dependent signaling pathway affected by R527H-mutated LMNA expression and possible targets of drug intervention. R527H-mutated LMNA fails to regulate TGFbeta 2 levels causing elevated levels of TGFbeta 2 in U2-OS cells. Downstream events are activation of the Akt pathway and ERK 1/2 activation, which influence mTORC1 activity. The Akt/mTOR pathway can be blocked at diverse levels using MK2206 to inhibit Akt, RAD001 or statins (mevinolin) to inhibit mTORC1. Mevinolin also acts on mutated prelamin A avoiding protein farnesylation and favoring phosphorylation by Akt and lysosomal degradation of prelamin A (4, 5). RAD001 is also expected to trigger prelamin A degradation (33). Anti-TGFbeta 2 neutralizing antibody can be used to block the whole signaling pathway.

Mentions: Several signaling effectors act downstream of TGFbeta receptor activation [32]. Here, we could demonstrate involvement of the Akt/mTOR pathway in TGFbeta 2-dependent OPG and cathepsin K increase by testing the effectors of that pathway in R527H U2-OS. The examined signaling molecules, including Akt, P70S6 kinase (P70S6K) and S6 ribosomal protein (S6RP) were not affected by expression of WT LMNA, but were significantly activated in R527H-LMNA transfected cells and in cells expressing farnesylated prelamin A (Figure 3A–3D). Neutralization of TGFbeta 2 activity inhibited the Akt/mTOR pathway in R527H LMNA U2-OS (Figure 3A–3D). Moreover, we observed a striking reduction of ERK 1/2 phosphorylation in cells overexpressing WT LMNA and unprocessable prelamin A, but not in R527H U2-OS (Figure 3E–3F). Activation of the MAPkinase-ERK 1/2 pathway is an effect previously described in mouse models of muscular laminopathies (13). Intriguingly, ERK 1/2 activity may converge on mTOR as does the Akt pathway (Figure 3G). These findings and other considerations reported below prompted us to block the Akt/mTOR pathway using several inhibitors (Figure 3G) and test the effect on OPG and cathepsin K modulation.


Modulation of TGFbeta 2 levels by lamin A in U2-OS osteoblast-like cells: understanding the osteolytic process triggered by altered lamins.

Evangelisti C, Bernasconi P, Cavalcante P, Cappelletti C, D'Apice MR, Sbraccia P, Novelli G, Prencipe S, Lemma S, Baldini N, Avnet S, Squarzoni S, Martelli AM, Lattanzi G - Oncotarget (2015)

The Akt/mTOR pathway is modulated by lamins through TGFbeta 2(A) Western blot analysis of U2-OS cells transfected with mock or WT, R527H or L647 LMNA plasmids showing the amount of active Akt (Thr308p-Akt and Ser473p-Akt), p70S6K (p-p70S6K) and S6RP (p-S6RP) and their total amount (Akt, p70S6K and S6RP, respectively). Actin bands show equal loading. The corresponding densitometric analysis of immunoblotted bands is reported in (B) as mean values of three different experiments +/– standard deviation. (C) Neutralization of TGFbeta 2 activity in R527H U2-OS cells blocks the activation of the Akt/mTOR pathway. U2-OS expressing WT or R527H LMNA were subjected to western blot analysis. R527H U2-OS were left untreated (R527H) or treated with anti-TGFbeta 2 neutralizing antibody (right lane, TGFbeta2 Ab R527H) as detailed in the methods section. The corresponding densitometric analysis of immunoblotted bands is reported in (D) as mean values of three different experiments +/– standard deviation. (E) Phosphorylated ERK 1/2 (p-ERK 1/2) and ERK 1/2 levels in U2-OS 24 hours after transfection of mock (CTR), WT, R527H or L647R LMNA plasmids. ERK1/2 activity is reduced by expression of WT LMNA, but not by R527 LMNA. Statistically significant differences (P < 0.05) are indicated by asterisks in (in B, D, (F)). (G) Schematic representation of the TGFbeta 2-dependent signaling pathway affected by R527H-mutated LMNA expression and possible targets of drug intervention. R527H-mutated LMNA fails to regulate TGFbeta 2 levels causing elevated levels of TGFbeta 2 in U2-OS cells. Downstream events are activation of the Akt pathway and ERK 1/2 activation, which influence mTORC1 activity. The Akt/mTOR pathway can be blocked at diverse levels using MK2206 to inhibit Akt, RAD001 or statins (mevinolin) to inhibit mTORC1. Mevinolin also acts on mutated prelamin A avoiding protein farnesylation and favoring phosphorylation by Akt and lysosomal degradation of prelamin A (4, 5). RAD001 is also expected to trigger prelamin A degradation (33). Anti-TGFbeta 2 neutralizing antibody can be used to block the whole signaling pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480690&req=5

Figure 3: The Akt/mTOR pathway is modulated by lamins through TGFbeta 2(A) Western blot analysis of U2-OS cells transfected with mock or WT, R527H or L647 LMNA plasmids showing the amount of active Akt (Thr308p-Akt and Ser473p-Akt), p70S6K (p-p70S6K) and S6RP (p-S6RP) and their total amount (Akt, p70S6K and S6RP, respectively). Actin bands show equal loading. The corresponding densitometric analysis of immunoblotted bands is reported in (B) as mean values of three different experiments +/– standard deviation. (C) Neutralization of TGFbeta 2 activity in R527H U2-OS cells blocks the activation of the Akt/mTOR pathway. U2-OS expressing WT or R527H LMNA were subjected to western blot analysis. R527H U2-OS were left untreated (R527H) or treated with anti-TGFbeta 2 neutralizing antibody (right lane, TGFbeta2 Ab R527H) as detailed in the methods section. The corresponding densitometric analysis of immunoblotted bands is reported in (D) as mean values of three different experiments +/– standard deviation. (E) Phosphorylated ERK 1/2 (p-ERK 1/2) and ERK 1/2 levels in U2-OS 24 hours after transfection of mock (CTR), WT, R527H or L647R LMNA plasmids. ERK1/2 activity is reduced by expression of WT LMNA, but not by R527 LMNA. Statistically significant differences (P < 0.05) are indicated by asterisks in (in B, D, (F)). (G) Schematic representation of the TGFbeta 2-dependent signaling pathway affected by R527H-mutated LMNA expression and possible targets of drug intervention. R527H-mutated LMNA fails to regulate TGFbeta 2 levels causing elevated levels of TGFbeta 2 in U2-OS cells. Downstream events are activation of the Akt pathway and ERK 1/2 activation, which influence mTORC1 activity. The Akt/mTOR pathway can be blocked at diverse levels using MK2206 to inhibit Akt, RAD001 or statins (mevinolin) to inhibit mTORC1. Mevinolin also acts on mutated prelamin A avoiding protein farnesylation and favoring phosphorylation by Akt and lysosomal degradation of prelamin A (4, 5). RAD001 is also expected to trigger prelamin A degradation (33). Anti-TGFbeta 2 neutralizing antibody can be used to block the whole signaling pathway.
Mentions: Several signaling effectors act downstream of TGFbeta receptor activation [32]. Here, we could demonstrate involvement of the Akt/mTOR pathway in TGFbeta 2-dependent OPG and cathepsin K increase by testing the effectors of that pathway in R527H U2-OS. The examined signaling molecules, including Akt, P70S6 kinase (P70S6K) and S6 ribosomal protein (S6RP) were not affected by expression of WT LMNA, but were significantly activated in R527H-LMNA transfected cells and in cells expressing farnesylated prelamin A (Figure 3A–3D). Neutralization of TGFbeta 2 activity inhibited the Akt/mTOR pathway in R527H LMNA U2-OS (Figure 3A–3D). Moreover, we observed a striking reduction of ERK 1/2 phosphorylation in cells overexpressing WT LMNA and unprocessable prelamin A, but not in R527H U2-OS (Figure 3E–3F). Activation of the MAPkinase-ERK 1/2 pathway is an effect previously described in mouse models of muscular laminopathies (13). Intriguingly, ERK 1/2 activity may converge on mTOR as does the Akt pathway (Figure 3G). These findings and other considerations reported below prompted us to block the Akt/mTOR pathway using several inhibitors (Figure 3G) and test the effect on OPG and cathepsin K modulation.

Bottom Line: Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2.TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment.Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

View Article: PubMed Central - PubMed

Affiliation: Rizzoli Orthopedic Institute, Laboratory of Musculoskeletal Cell Biology, CNR Institute for Molecular Genetics, Unit of Bologna, Bologna, Italy.

ABSTRACT
Transforming growth factor beta (TGFbeta) plays an essential role in bone homeostasis and deregulation of TGFbeta occurs in bone pathologies. Patients affected by Mandibuloacral Dysplasia (MADA), a progeroid disease linked to LMNA mutations, suffer from an osteolytic process. Our previous work showed that MADA osteoblasts secrete excess amount of TGFbeta 2, which in turn elicits differentiation of human blood precursors into osteoclasts. Here, we sought to determine how altered lamin A affects TGFbeta signaling. Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2. TGFbeta 2 in turn, triggers the Akt/mTOR pathway and upregulates osteoprotegerin and cathepsin K. TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment. Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

No MeSH data available.


Related in: MedlinePlus