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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

RAD001 treatment of R527H LMNA rescues the Akt/mTOR signaling pathway, and OPG levels and reduces cathepsin K amount(A) Western blot analysis of the Akt/mTOR signaling pathway effectors in U2-OS cells transfected with WT LMNA (left panels) or R527H LMNA (right panels). Cells were subjected to Akt inhibitor MK2206 or mTORC1 inhibitor RAD001. The immunoblotted bands correspond to active Akt (Thr308p-Akt and Ser473p-Akt), total Akt (Akt), active p70S6K (p-p70S6K) and active S6RP (p-S6RP) and total p70S6K and S6RP. Actin bands show equal loading. (B) Western blot analysis of prelamin A, TGFbeta 2, OPG and cathepsin K in the samples analyzed in (A) The densitometric analysis of immunoblotted samples shown in (B) is reported in (C) as mean values of three different experiments +/– standard deviation. Statistically significant values relative to each untreated sample (CTR), are indicated by asterisks.
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Figure 4: RAD001 treatment of R527H LMNA rescues the Akt/mTOR signaling pathway, and OPG levels and reduces cathepsin K amount(A) Western blot analysis of the Akt/mTOR signaling pathway effectors in U2-OS cells transfected with WT LMNA (left panels) or R527H LMNA (right panels). Cells were subjected to Akt inhibitor MK2206 or mTORC1 inhibitor RAD001. The immunoblotted bands correspond to active Akt (Thr308p-Akt and Ser473p-Akt), total Akt (Akt), active p70S6K (p-p70S6K) and active S6RP (p-S6RP) and total p70S6K and S6RP. Actin bands show equal loading. (B) Western blot analysis of prelamin A, TGFbeta 2, OPG and cathepsin K in the samples analyzed in (A) The densitometric analysis of immunoblotted samples shown in (B) is reported in (C) as mean values of three different experiments +/– standard deviation. Statistically significant values relative to each untreated sample (CTR), are indicated by asterisks.

Mentions: To support the above reported involvement of Akt/mTOR pathway in the altered regulation of OPG and cathepsin K, we inhibited Akt activity, using MK2206 and mTOR, using the rapamycin analog RAD001 that impairs mTORC1 activity (Figure 4). While Akt activity was completely inhibited by MK2206, protein amount was increased (Figure 4A). Akt inhibition caused dysregulation of its downstream effectors, including P70S6K and S6RP, although with different effects in WT lamin A versus R527H-mutated lamin A-transfected cells (Figure 4A). Phosphorylation of P70S6K was increased in WT-transfected cells and decreased in R527H-transfected U2-OS, while S6RP phosphorylation was consistently inhibited in all MK2206-treated cells (Figure 4A). RAD001 minimally affected Akt activity, but elicited protein increase (Figure 4A), possibly due to a feedback mechanism. Complete inhibition of P70S6K and S6RP phosphorylation was observed in all RAD001-treated cells (Figure 4A).


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)

RAD001 treatment of R527H LMNA rescues the Akt/mTOR signaling pathway, and OPG levels and reduces cathepsin K amount(A) Western blot analysis of the Akt/mTOR signaling pathway effectors in U2-OS cells transfected with WT LMNA (left panels) or R527H LMNA (right panels). Cells were subjected to Akt inhibitor MK2206 or mTORC1 inhibitor RAD001. The immunoblotted bands correspond to active Akt (Thr308p-Akt and Ser473p-Akt), total Akt (Akt), active p70S6K (p-p70S6K) and active S6RP (p-S6RP) and total p70S6K and S6RP. Actin bands show equal loading. (B) Western blot analysis of prelamin A, TGFbeta 2, OPG and cathepsin K in the samples analyzed in (A) The densitometric analysis of immunoblotted samples shown in (B) is reported in (C) as mean values of three different experiments +/– standard deviation. Statistically significant values relative to each untreated sample (CTR), are indicated by asterisks.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: RAD001 treatment of R527H LMNA rescues the Akt/mTOR signaling pathway, and OPG levels and reduces cathepsin K amount(A) Western blot analysis of the Akt/mTOR signaling pathway effectors in U2-OS cells transfected with WT LMNA (left panels) or R527H LMNA (right panels). Cells were subjected to Akt inhibitor MK2206 or mTORC1 inhibitor RAD001. The immunoblotted bands correspond to active Akt (Thr308p-Akt and Ser473p-Akt), total Akt (Akt), active p70S6K (p-p70S6K) and active S6RP (p-S6RP) and total p70S6K and S6RP. Actin bands show equal loading. (B) Western blot analysis of prelamin A, TGFbeta 2, OPG and cathepsin K in the samples analyzed in (A) The densitometric analysis of immunoblotted samples shown in (B) is reported in (C) as mean values of three different experiments +/– standard deviation. Statistically significant values relative to each untreated sample (CTR), are indicated by asterisks.
Mentions: To support the above reported involvement of Akt/mTOR pathway in the altered regulation of OPG and cathepsin K, we inhibited Akt activity, using MK2206 and mTOR, using the rapamycin analog RAD001 that impairs mTORC1 activity (Figure 4). While Akt activity was completely inhibited by MK2206, protein amount was increased (Figure 4A). Akt inhibition caused dysregulation of its downstream effectors, including P70S6K and S6RP, although with different effects in WT lamin A versus R527H-mutated lamin A-transfected cells (Figure 4A). Phosphorylation of P70S6K was increased in WT-transfected cells and decreased in R527H-transfected U2-OS, while S6RP phosphorylation was consistently inhibited in all MK2206-treated cells (Figure 4A). RAD001 minimally affected Akt activity, but elicited protein increase (Figure 4A), possibly due to a feedback mechanism. Complete inhibition of P70S6K and S6RP phosphorylation was observed in all RAD001-treated cells (Figure 4A).

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