Wang, Shuo (2018-05). The Potential Role of Erk1/2 Map Kinases in the Regulation of Neuromuscular Junction Integrity and Bone Morphogenetic Protein Signaling. Doctoral Dissertation.
Thesis
It is known that extracellular signal-regulated kinases 1 and 2 (ERK1/2) play important roles in development and maintenance of skeletal muscles in vitro and in vivo. However, it is not clear whether muscle ERK1/2 can regulate neuromuscular junction (NMJ) structure and function and (or) have unique substrates and downstream pathways in skeletal muscle. Previously, we generated an ERK 1/2 double knockout (DKO) mouse line using germ line Erk1 mutation and Cre-loxP deletion of Erk2. Those DKO animals were viable after birth but displayed stunted postnatal growth, muscle weakness, and shorter lifespan. Next, two typical fast-twitch muscles, the sternomastoid (STN) and the tibialis anterior (TA), were examined. A mixture of fiber loss and mild muscle atrophy was found, and STN but not TA underwent partial denervation. To explore the role of ERK1/2 in slow-twitch, type 1 muscle fibers, we studied the mutant soleus muscle (SOL) in the aspect of morphology, expression of denervation and synaptic markers, and mitochondrial function. Next, we examined the status of bone morphogenetic (BMP) signaling in mutant SOL and myotubes by measuring mRNA and phosphorylation level of several BMP components. It is shown that young adult mutant SOL was drastically wasted, with highly atrophied type 1 fibers, denervation at most synaptic sites, induction of "fetal" acetylcholine receptor (AChR?) subunit, reduction of "adult" AChR? subunit, and impaired mitochondrial function. In weanlings, fiber morphology and mitochondrial markers were normal, yet AChR? upregulation and AChR? downregulation were observed. In mutant weanlings, most of the fetal AChRs appeared at NMJs on type1 muscle fibers. These results suggest that: (1) ERK1/2 are critical for slow-twitch fiber growth; (2) a defective ?/?-AChR subunit switch, preferentially at synapses on slow fibers, precedes denervation and wasting of mutant SOL; (3) the neuromuscular synapse is a primary subcellular target for muscle ERK1/2 function in vivo. It is shown that mutant SOL exhibited alterations in the BMP pathway: decreased expression of Bmpr1b, unchanged expression of the target gene Id-1, the negative regulator Noggin and the intracellular kinase Smad6, and unchanged phosphorylation level of Smad1/5/8. These experiments suggest that unlike normal muscle, BMP signaling is not activated in mutant SOL after denervation. However, Bmpr1b expression and Smad1/5/8 phosphorylation were not altered in BMP-treated myotubes subjected to pharmacological inhibition of ERK1/2 activation. These results suggest that ERK1/2 regulate BMP signaling in SOL through an indirect mechanism.
