By Jamshed Arslan Pharm.D.
Our upright posture and balance depend on a jelly-like material, called nucleus pulposus (NP), in the middle of intervertebral discs. NP cells protect us from disc degeneration by maintaining optimal amounts of proteoglycans (proteins bonded to glycosaminoglycans) in the NP matrix. This process can be facilitated by TGF-beta, which stimulates the synthesis of sulfated glycosaminoglycan (sGAG) and chondroitin sulfate proteoglycan 1 in the NP cells. The synthesis of sGAG depends on chondroitin polymerizing factor (ChPF), an enzyme that extends the chondroitin sulfate (CS) backbone in sGAG. However, the effects of TGF-beta on ChPF and the signaling pathways involved in the TGF-beta-mediated activity in the NP cells largely remained unclear.
Now, a team of researchers from Changzheng Hospital and the Second Afﬁliated Hospital of Harbin Medical University, China, have found that TGF-beta upregulates ChPF, and consequently leads to sGAG synthesis in the NP cells. They found that RhoA /ROCK1, Smad3, p38, JNK, and ERK1/2 signaling pathways are involved in this process.
Immunocytochemistry/Immunofluorescence: CHSS2 Antibody [NBP1-88087] - Immunofluorescent staining of human cell line U-251 MG shows localization to cytosol, centrosome & vesicles.
Negative correlation of ChPF with disc disease
The researchers first set out to find a correlation between ChPF expression in human NP tissue samples and degenerative intervertebral disc disease. They found that increased disease severity, based on MRI scans, could be correlated with lower ChPF expression at mRNA and protein levels. This meant that ChPF is negatively correlated with disc disease.
TGF-beta upregulates ChPF
To determine the role of TGF-beta in ChPF expression, NP cells isolated from rats were treated with TGF-beta. A dose-dependent increase in the ChPF mRNA and protein was reported. To confirm these results, NP cells were pretreated with an inhibitor of TGF-beta receptor kinase, which abolished ChPF upregulation. After discovering that TGF-beta upregulates ChPF in the NP cells, their next step was to study the response of downstream effectors of TGF-beta, including Smad2/3, small GTPase RhoA, ROCK1, ROCK2, JNK, and p38.
Signaling pathways involved in TGF-beta-induced upregulation of ChPF
- Smad3-Western blot of rat NP cells after TGF-beta stimulation showed a time- and dose-dependent increase in phosphorylated (p-)Smad2 and p-Smad3. However, it was the knockdown of Smad3 and not Smad2 by shRNA that inhibited TGF-beta-induced ChPF expression and sGAG synthesis.
- RhoA/ROCK1-Similar to Smad2/3 induction, RhoA activation depended on TGF-beta concentration. Western blotting revealed a time- and dose-dependent accumulation of p-ROCK1 p-ROCK2, ROCK1, and ROCK2, which are downstream effectors of RhoA. However, real-time PCR and western blot analyses showed that TGF-beta-mediated ChPF and sGAG production were inhibited by blocking RhoA and ROCK1, but not ROCK2.
- MAPK signaling pathways-Within an hour of TGF-beta treatment, NP cells showed increased phosphorylation of p38, JNK, and ERK1/2, which persisted even when Smad3 was knocked down by shRNA. Inhibitors of RhoA and ROCK1 compromised only the JNK activation, but not p38 or ERK1/2 signaling. Furthermore, blocking any of the three MAPK signaling pathways (by using chemical inhibitors of p38, JNK, and ERK1/2) was found to compromise the TGF-beta-mediated production of ChPF and sGAG in the rat NP cells.
This study has both diagnostic and therapeutic importance. The team has shown a diagnostic importance of ChPF expression in grading the severity of degenerative disc disease. In light of this research, delivery of TGF-beta to NP may provide relief to the patients and the population at risk.
Explore TGF-beta Poster
Jamshed Arslan, Pharm D.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans
and transgenic mice.
Hu, Bo, et al. “TGF-bStimulates Expression of Chondroitin Polymerizing Factor in Nucleus Pulposus Cells Through the Smad3, RhoA/ROCK1, and MAPK Signaling Pathways.” Journal of Cellular Biochemistry, vol. 119, no. 1, 2018, pp. 566–579. doi: 10.1002/jcb.26215.