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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

Cytokine secretion in U2-OS osteoblast-like cells is influenced by LMNA expressionCell culture media mock-transfected U2-OS (CTR), or U2-OS transfected with WT, L647R or R527H LMNA plasmids were subjected to multiplex cytokine assay. Results for 28 cytokines/growth factors are reported in the graphs. Media were collected 24 or 72 hours after transfection. Protein values indicated on the Y axes are reported as pg/ml. Graphs are representative of three different experiments.
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Figure 1: Cytokine secretion in U2-OS osteoblast-like cells is influenced by LMNA expressionCell culture media mock-transfected U2-OS (CTR), or U2-OS transfected with WT, L647R or R527H LMNA plasmids were subjected to multiplex cytokine assay. Results for 28 cytokines/growth factors are reported in the graphs. Media were collected 24 or 72 hours after transfection. Protein values indicated on the Y axes are reported as pg/ml. Graphs are representative of three different experiments.

Mentions: To get insights into the effect of lamins on TGFbeta 2 regulation, U2-OS cells were transfected with FLAG-tagged plasmids expressing wild-type prelamin A (WT), which is produced as mature lamin A, or uncleavable prelamin A (L647R), which yields accumulation of farnesylated prelamin A. Moreover, to investigate the molecular pathway triggering altered cytokine regulation in MADA osteoblasts, we introduced the R527H LMNA mutant in U2-OS. At first, to test the secretory profile of transfected U2-OS cells, we examined conditioned medium from those cell cultures by multiplex cytokine assay (Figure 1). Our data showed a general effect of LMNA expression on the secretory profile of osteoblast-like cells and pointed to an inhibitory effect for most cytokines and growth factors including TGFbeta 1 and 3 (Figure 1) and TGFbeta 2 (Figure 2A). Only in the case of Mip-1a and b and RANTES (CCL3) lamin A overexpression elicited chemokine increase (Figure 1), an interesting finding based on the role of these molecules in osteolytic processes [30]. In most cases, overexpression of R527H LMNA or farnesylated prelamin A accumulation (L647R LMNA) elicited the same effect on cytokine secretion as wild-type lamin A (Figure 1).


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)

Cytokine secretion in U2-OS osteoblast-like cells is influenced by LMNA expressionCell culture media mock-transfected U2-OS (CTR), or U2-OS transfected with WT, L647R or R527H LMNA plasmids were subjected to multiplex cytokine assay. Results for 28 cytokines/growth factors are reported in the graphs. Media were collected 24 or 72 hours after transfection. Protein values indicated on the Y axes are reported as pg/ml. Graphs are representative of three different experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Cytokine secretion in U2-OS osteoblast-like cells is influenced by LMNA expressionCell culture media mock-transfected U2-OS (CTR), or U2-OS transfected with WT, L647R or R527H LMNA plasmids were subjected to multiplex cytokine assay. Results for 28 cytokines/growth factors are reported in the graphs. Media were collected 24 or 72 hours after transfection. Protein values indicated on the Y axes are reported as pg/ml. Graphs are representative of three different experiments.
Mentions: To get insights into the effect of lamins on TGFbeta 2 regulation, U2-OS cells were transfected with FLAG-tagged plasmids expressing wild-type prelamin A (WT), which is produced as mature lamin A, or uncleavable prelamin A (L647R), which yields accumulation of farnesylated prelamin A. Moreover, to investigate the molecular pathway triggering altered cytokine regulation in MADA osteoblasts, we introduced the R527H LMNA mutant in U2-OS. At first, to test the secretory profile of transfected U2-OS cells, we examined conditioned medium from those cell cultures by multiplex cytokine assay (Figure 1). Our data showed a general effect of LMNA expression on the secretory profile of osteoblast-like cells and pointed to an inhibitory effect for most cytokines and growth factors including TGFbeta 1 and 3 (Figure 1) and TGFbeta 2 (Figure 2A). Only in the case of Mip-1a and b and RANTES (CCL3) lamin A overexpression elicited chemokine increase (Figure 1), an interesting finding based on the role of these molecules in osteolytic processes [30]. In most cases, overexpression of R527H LMNA or farnesylated prelamin A accumulation (L647R LMNA) elicited the same effect on cytokine secretion as wild-type lamin A (Figure 1).

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