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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

Reduced number of TRACP-positive multinuclear cells (osteoclasts) in human peripheral blood monocyte cultures subjected to conditioned medium from RAD001- or mevinolin-treated R527H LMNA U2-OS cells(A) Human mononuclear cells were induced to differentiate into osteoclasts for 8 days by adding medium from untreated WT LMNA, R527H LMNA U2-OS, or R527H LMNA U2-OS treated with RAD001 (72 hours) or mevinolin (24 hours). TRACP expression is revealed by red precipitates in representative pictures, nuclei are counterstained with Hoechst 33258 (Bar, 20 μm). Arrowheads indicate osteoclasts and their nuclei. (B) Quantitative analysis of osteoclast formation. Osteoclast number per sample is reported on the Y axis. Analysis was performed on six countings of the TRACP positive cells that showed three or more than three nuclei. Data are expressed as mean+/− standard deviation. Statistically significant differences (p < 0.05) are indicated by asterisks.
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Figure 6: Reduced number of TRACP-positive multinuclear cells (osteoclasts) in human peripheral blood monocyte cultures subjected to conditioned medium from RAD001- or mevinolin-treated R527H LMNA U2-OS cells(A) Human mononuclear cells were induced to differentiate into osteoclasts for 8 days by adding medium from untreated WT LMNA, R527H LMNA U2-OS, or R527H LMNA U2-OS treated with RAD001 (72 hours) or mevinolin (24 hours). TRACP expression is revealed by red precipitates in representative pictures, nuclei are counterstained with Hoechst 33258 (Bar, 20 μm). Arrowheads indicate osteoclasts and their nuclei. (B) Quantitative analysis of osteoclast formation. Osteoclast number per sample is reported on the Y axis. Analysis was performed on six countings of the TRACP positive cells that showed three or more than three nuclei. Data are expressed as mean+/− standard deviation. Statistically significant differences (p < 0.05) are indicated by asterisks.

Mentions: To test the efficacy of RAD001 or mevinolin treatment in terms of inhibition of osteoclastogenesis, we used conditioned media from R527H U2-OS cells to trigger osteoclast differentiation in peripheral blood monocytes from healthy donors. As shown in Figure 6, R527H U2-OS-conditioned precursors differentiated more efficiently than WT U2-OS-conditioned monocytes. However, using conditioned medium from RAD001-treated R527H cells, osteoclast differentiation was significantly reduced (Figure 6A and 6B).


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)

Reduced number of TRACP-positive multinuclear cells (osteoclasts) in human peripheral blood monocyte cultures subjected to conditioned medium from RAD001- or mevinolin-treated R527H LMNA U2-OS cells(A) Human mononuclear cells were induced to differentiate into osteoclasts for 8 days by adding medium from untreated WT LMNA, R527H LMNA U2-OS, or R527H LMNA U2-OS treated with RAD001 (72 hours) or mevinolin (24 hours). TRACP expression is revealed by red precipitates in representative pictures, nuclei are counterstained with Hoechst 33258 (Bar, 20 μm). Arrowheads indicate osteoclasts and their nuclei. (B) Quantitative analysis of osteoclast formation. Osteoclast number per sample is reported on the Y axis. Analysis was performed on six countings of the TRACP positive cells that showed three or more than three nuclei. Data are expressed as mean+/− standard deviation. Statistically significant differences (p < 0.05) are indicated by asterisks.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Reduced number of TRACP-positive multinuclear cells (osteoclasts) in human peripheral blood monocyte cultures subjected to conditioned medium from RAD001- or mevinolin-treated R527H LMNA U2-OS cells(A) Human mononuclear cells were induced to differentiate into osteoclasts for 8 days by adding medium from untreated WT LMNA, R527H LMNA U2-OS, or R527H LMNA U2-OS treated with RAD001 (72 hours) or mevinolin (24 hours). TRACP expression is revealed by red precipitates in representative pictures, nuclei are counterstained with Hoechst 33258 (Bar, 20 μm). Arrowheads indicate osteoclasts and their nuclei. (B) Quantitative analysis of osteoclast formation. Osteoclast number per sample is reported on the Y axis. Analysis was performed on six countings of the TRACP positive cells that showed three or more than three nuclei. Data are expressed as mean+/− standard deviation. Statistically significant differences (p < 0.05) are indicated by asterisks.
Mentions: To test the efficacy of RAD001 or mevinolin treatment in terms of inhibition of osteoclastogenesis, we used conditioned media from R527H U2-OS cells to trigger osteoclast differentiation in peripheral blood monocytes from healthy donors. As shown in Figure 6, R527H U2-OS-conditioned precursors differentiated more efficiently than WT U2-OS-conditioned monocytes. However, using conditioned medium from RAD001-treated R527H cells, osteoclast differentiation was significantly reduced (Figure 6A and 6B).

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