Limits...
Transforming Growth Factor Beta (TGF-β) Is a Muscle Biomarker of Disease Progression in ALS and Correlates with Smad Expression.

Si Y, Kim S, Cui X, Zheng L, Oh SJ, Anderson T, AlSharabati M, Kazamel M, Volpicelli-Daley L, Bamman MM, Yu S, King PH - PLoS ONE (2015)

Bottom Line: TGF-β1, 2 and 3 mRNAs were increased in ALS muscle samples compared to controls and correlated with muscle strength and Smads1, 2, 5 and 8.In conclusion, TGF-β1, 2 and 3 are novel biomarkers of ALS in skeletal muscle.These ligands are capable of upregulating and activating Smads and thus may contribute to the Smad signaling pathway in ALS muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Birmingham VA Medical Center, Birmingham, Alabama, United States of America.

ABSTRACT
We recently identified Smads1, 5 and 8 as muscle biomarkers in human ALS. In the ALS mouse, these markers are elevated and track disease progression. Smads are signal transducers and become activated upon receptor engagement of ligands from the TGF-β superfamily. Here, we sought to characterize ligands linked to activation of Smads in ALS muscle and their role as biomarkers of disease progression. RNA sequencing data of ALS muscle samples were mined for TGF-β superfamily ligands. Candidate targets were validated by qRT-PCR in a large cohort of human ALS muscle biopsy samples and in the G93A SOD1 mouse. Protein expression was evaluated by Western blot, ELISA and immunohistochemistry. C2C12 muscle cells were used to assess Smad activation and induction. TGF-β1, 2 and 3 mRNAs were increased in ALS muscle samples compared to controls and correlated with muscle strength and Smads1, 2, 5 and 8. In the G93A SOD1 mouse, the temporal pattern of TGF-β expression paralleled the Smads and increased with disease progression. TGF-β1 immunoreactivity was detected in mononuclear cells surrounding muscle fibers in ALS samples. In muscle cells, TGF-β ligands were capable of activating Smads. In conclusion, TGF-β1, 2 and 3 are novel biomarkers of ALS in skeletal muscle. Their correlation with weakness in human ALS and their progressive increase with advancing disease in the ALS mouse suggest that they, as with the Smads, can track disease progression. These ligands are capable of upregulating and activating Smads and thus may contribute to the Smad signaling pathway in ALS muscle.

No MeSH data available.


Related in: MedlinePlus

TGF-β mRNA levels are increased at early stages of ALS in the G93A SOD1 mouse.Total RNA was isolated from G93A SOD1 mice and littermate controls (WT) at 40, 60 and 105 d (preclinical stages as previously defined [4]), and early and late clinical stages (125 and 150 d). Samples were analyzed by qRT-PCR for TGF-β1, 2, 3 and BMP4 mRNA expression. Data points represent the mean ± SE of 6–8 mice. * p < 0.05; ** < 0.005; **** < 0.0001. RQ, relative quantity.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4574401&req=5

pone.0138425.g003: TGF-β mRNA levels are increased at early stages of ALS in the G93A SOD1 mouse.Total RNA was isolated from G93A SOD1 mice and littermate controls (WT) at 40, 60 and 105 d (preclinical stages as previously defined [4]), and early and late clinical stages (125 and 150 d). Samples were analyzed by qRT-PCR for TGF-β1, 2, 3 and BMP4 mRNA expression. Data points represent the mean ± SE of 6–8 mice. * p < 0.05; ** < 0.005; **** < 0.0001. RQ, relative quantity.

Mentions: We next determined whether TGF-β mRNA upregulation occurred in skeletal muscle from the G93A SOD1 mouse and if the temporal pattern of expression paralleled that of the Smads. In our previous report, we observed induction of Smad1, 5 and 8 mRNAs between post-natal day 40 and 60 [4]. At that time interval, no overt clinical manifestations are observed, but subtle motor deficits have been described [9]. In our colony, the animals have a longer survival time (mean of 161 days) and no gender effect (data not shown). These features are similar to what has previously been reported for this model [9]. Further details regarding progression and disease onset are provided elsewhere [4]. For all three TGF-β isoforms we observed a significant increase in mRNA compared to littermate controls starting at 60 d and throughout the clinical course (Fig 3). TGF-β1 showed the greatest increase at each time point (more than 15-fold by end-stage). There was no increase at 40 d in any of the isoforms indicating a temporal pattern similar to the Smads [4]. BMP4, another TGF-β receptor ligand which showed a non-significant upward trend in our RNA sequencing analysis, increased only in the later stages (125 and 150 d) indicating that TGF-β1, 2 and 3 mRNA induction was relatively selective in the early stages of disease. We next assessed protein expression. For TGF-β1, we observed gradual increases by ELISA with disease progression (Fig 4A). Detection required acid activation indicating that the ligand is predominantly in the latent form [10]. Western blot (under reducing conditions) showed a similar increase in the mature form of TGF-β1 with disease progression. Immunohistochemistry with a TGF-β1 antibody showed labeling of mononuclear cells adjacent to muscle borders as outlined by WGA staining (Fig 4B). Little to no staining was observed in WT muscle. For TGF-β2 and 3, Western blot analysis showed an increase of pre-processed protein in mutant mice over control at each age toward end-stage. Densitometry of three independent mouse samples indicated a 2–3-fold increase over age-matched controls, with an upward trend at end-stage (Fig 4C). Processed forms were not detected, and immunohistochemistry did not show consistent staining for either isoform (not shown).


Transforming Growth Factor Beta (TGF-β) Is a Muscle Biomarker of Disease Progression in ALS and Correlates with Smad Expression.

Si Y, Kim S, Cui X, Zheng L, Oh SJ, Anderson T, AlSharabati M, Kazamel M, Volpicelli-Daley L, Bamman MM, Yu S, King PH - PLoS ONE (2015)

TGF-β mRNA levels are increased at early stages of ALS in the G93A SOD1 mouse.Total RNA was isolated from G93A SOD1 mice and littermate controls (WT) at 40, 60 and 105 d (preclinical stages as previously defined [4]), and early and late clinical stages (125 and 150 d). Samples were analyzed by qRT-PCR for TGF-β1, 2, 3 and BMP4 mRNA expression. Data points represent the mean ± SE of 6–8 mice. * p < 0.05; ** < 0.005; **** < 0.0001. RQ, relative quantity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4574401&req=5

pone.0138425.g003: TGF-β mRNA levels are increased at early stages of ALS in the G93A SOD1 mouse.Total RNA was isolated from G93A SOD1 mice and littermate controls (WT) at 40, 60 and 105 d (preclinical stages as previously defined [4]), and early and late clinical stages (125 and 150 d). Samples were analyzed by qRT-PCR for TGF-β1, 2, 3 and BMP4 mRNA expression. Data points represent the mean ± SE of 6–8 mice. * p < 0.05; ** < 0.005; **** < 0.0001. RQ, relative quantity.
Mentions: We next determined whether TGF-β mRNA upregulation occurred in skeletal muscle from the G93A SOD1 mouse and if the temporal pattern of expression paralleled that of the Smads. In our previous report, we observed induction of Smad1, 5 and 8 mRNAs between post-natal day 40 and 60 [4]. At that time interval, no overt clinical manifestations are observed, but subtle motor deficits have been described [9]. In our colony, the animals have a longer survival time (mean of 161 days) and no gender effect (data not shown). These features are similar to what has previously been reported for this model [9]. Further details regarding progression and disease onset are provided elsewhere [4]. For all three TGF-β isoforms we observed a significant increase in mRNA compared to littermate controls starting at 60 d and throughout the clinical course (Fig 3). TGF-β1 showed the greatest increase at each time point (more than 15-fold by end-stage). There was no increase at 40 d in any of the isoforms indicating a temporal pattern similar to the Smads [4]. BMP4, another TGF-β receptor ligand which showed a non-significant upward trend in our RNA sequencing analysis, increased only in the later stages (125 and 150 d) indicating that TGF-β1, 2 and 3 mRNA induction was relatively selective in the early stages of disease. We next assessed protein expression. For TGF-β1, we observed gradual increases by ELISA with disease progression (Fig 4A). Detection required acid activation indicating that the ligand is predominantly in the latent form [10]. Western blot (under reducing conditions) showed a similar increase in the mature form of TGF-β1 with disease progression. Immunohistochemistry with a TGF-β1 antibody showed labeling of mononuclear cells adjacent to muscle borders as outlined by WGA staining (Fig 4B). Little to no staining was observed in WT muscle. For TGF-β2 and 3, Western blot analysis showed an increase of pre-processed protein in mutant mice over control at each age toward end-stage. Densitometry of three independent mouse samples indicated a 2–3-fold increase over age-matched controls, with an upward trend at end-stage (Fig 4C). Processed forms were not detected, and immunohistochemistry did not show consistent staining for either isoform (not shown).

Bottom Line: TGF-β1, 2 and 3 mRNAs were increased in ALS muscle samples compared to controls and correlated with muscle strength and Smads1, 2, 5 and 8.In conclusion, TGF-β1, 2 and 3 are novel biomarkers of ALS in skeletal muscle.These ligands are capable of upregulating and activating Smads and thus may contribute to the Smad signaling pathway in ALS muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Birmingham VA Medical Center, Birmingham, Alabama, United States of America.

ABSTRACT
We recently identified Smads1, 5 and 8 as muscle biomarkers in human ALS. In the ALS mouse, these markers are elevated and track disease progression. Smads are signal transducers and become activated upon receptor engagement of ligands from the TGF-β superfamily. Here, we sought to characterize ligands linked to activation of Smads in ALS muscle and their role as biomarkers of disease progression. RNA sequencing data of ALS muscle samples were mined for TGF-β superfamily ligands. Candidate targets were validated by qRT-PCR in a large cohort of human ALS muscle biopsy samples and in the G93A SOD1 mouse. Protein expression was evaluated by Western blot, ELISA and immunohistochemistry. C2C12 muscle cells were used to assess Smad activation and induction. TGF-β1, 2 and 3 mRNAs were increased in ALS muscle samples compared to controls and correlated with muscle strength and Smads1, 2, 5 and 8. In the G93A SOD1 mouse, the temporal pattern of TGF-β expression paralleled the Smads and increased with disease progression. TGF-β1 immunoreactivity was detected in mononuclear cells surrounding muscle fibers in ALS samples. In muscle cells, TGF-β ligands were capable of activating Smads. In conclusion, TGF-β1, 2 and 3 are novel biomarkers of ALS in skeletal muscle. Their correlation with weakness in human ALS and their progressive increase with advancing disease in the ALS mouse suggest that they, as with the Smads, can track disease progression. These ligands are capable of upregulating and activating Smads and thus may contribute to the Smad signaling pathway in ALS muscle.

No MeSH data available.


Related in: MedlinePlus