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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

Mevinolin treatment reduces TGFbeta 2 levels and attenuates Akt/mTOR signaling, OPG and cathepsin K levels in R527H LMNA U2-OS cells. U2-OS cells transfected with WT or R527H LMNA were left untreated or subjected to 24 hours mevinolin treatment (+MEV)(A) Western blot analysis of FLAG-lamin A, TGFbeta 2, OPG and cathepsin K (B) Densitometric analysis of immunoblotted proteins shown in (A). (C) Western blot analysis of 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 in (A) and (B). (D) Densitometric analysis of immunoblotted proteins shown in (C). Values in (B) and (D) are means +/− standard deviation of three values reported in different experiments. Statistically significant differences ( p < 0.05) relative to each untreated sample (WT or R527H), are indicated by asterisks.
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Figure 5: Mevinolin treatment reduces TGFbeta 2 levels and attenuates Akt/mTOR signaling, OPG and cathepsin K levels in R527H LMNA U2-OS cells. U2-OS cells transfected with WT or R527H LMNA were left untreated or subjected to 24 hours mevinolin treatment (+MEV)(A) Western blot analysis of FLAG-lamin A, TGFbeta 2, OPG and cathepsin K (B) Densitometric analysis of immunoblotted proteins shown in (A). (C) Western blot analysis of 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 in (A) and (B). (D) Densitometric analysis of immunoblotted proteins shown in (C). Values in (B) and (D) are means +/− standard deviation of three values reported in different experiments. Statistically significant differences ( p < 0.05) relative to each untreated sample (WT or R527H), are indicated by asterisks.

Mentions: U2-OS cells were transfected with WT- and R527H-LMNA plasmids and treated with mevinolin for 24 hours. Western blot analysis showed that in mevinolin-treated R527H U2-OS, TGFbeta 2, OPG and cathepsin K levels were significantly lowered (Figure 5A–5B).


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)

Mevinolin treatment reduces TGFbeta 2 levels and attenuates Akt/mTOR signaling, OPG and cathepsin K levels in R527H LMNA U2-OS cells. U2-OS cells transfected with WT or R527H LMNA were left untreated or subjected to 24 hours mevinolin treatment (+MEV)(A) Western blot analysis of FLAG-lamin A, TGFbeta 2, OPG and cathepsin K (B) Densitometric analysis of immunoblotted proteins shown in (A). (C) Western blot analysis of 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 in (A) and (B). (D) Densitometric analysis of immunoblotted proteins shown in (C). Values in (B) and (D) are means +/− standard deviation of three values reported in different experiments. Statistically significant differences ( p < 0.05) relative to each untreated sample (WT or R527H), are indicated by asterisks.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Mevinolin treatment reduces TGFbeta 2 levels and attenuates Akt/mTOR signaling, OPG and cathepsin K levels in R527H LMNA U2-OS cells. U2-OS cells transfected with WT or R527H LMNA were left untreated or subjected to 24 hours mevinolin treatment (+MEV)(A) Western blot analysis of FLAG-lamin A, TGFbeta 2, OPG and cathepsin K (B) Densitometric analysis of immunoblotted proteins shown in (A). (C) Western blot analysis of 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 in (A) and (B). (D) Densitometric analysis of immunoblotted proteins shown in (C). Values in (B) and (D) are means +/− standard deviation of three values reported in different experiments. Statistically significant differences ( p < 0.05) relative to each untreated sample (WT or R527H), are indicated by asterisks.
Mentions: U2-OS cells were transfected with WT- and R527H-LMNA plasmids and treated with mevinolin for 24 hours. Western blot analysis showed that in mevinolin-treated R527H U2-OS, TGFbeta 2, OPG and cathepsin K levels were significantly lowered (Figure 5A–5B).

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