A tale of two niches: differential functions for VCAM-1 in satellite cells under basal and injured conditions.

Cell-cell adhesion molecules play key roles in maintaining quiescence or promoting activation of various stem cells in their niche. Muscle stem cells called satellite cells (SC) are critical for skeletal muscle regeneration after injury, but little is known about the role of adhesion molecules in regulating the behavior of these stem cells. Vascular cell adhesion molecule-1 (VCAM-1) is a cell-cell adhesion protein expressed on quiescent and activated SC whose function is unknown in this context. We deleted Vcam1 from SC using an inducible Cre recombinase in young mice. In the injured niche, Vcam1-/- SC underwent premature lineage progression to a more differentiated state as well as apoptosis leading to a transient delay in myofiber growth during regeneration. Apoptosis was also increased in Vcam1-/- SC in vitro concomitant with decreased levels of phosphorylated Akt, a prosurvival signal activated by VCAM-1 signaling in other cell types. During muscle regeneration, we observed an influx of immune cells expressing α4 integrin, a component of the major, high-affinity VCAM-1 ligand, α4ß1 integrin. Furthermore, α4 integrin mRNA and protein were induced in SC 2 days after injury. These results suggest that SC interact with other SC as well as immune cells through α4ß1 integrin in the injured niche to promote expansion of SC. In the uninjured niche, multiple cell types also expressed α4 integrin. However, only basal fusion of Vcam1-/- SC with myofibers was decreased, contributing to decreased myofiber growth. These studies define differential roles for VCAM-1 in SC depending on the state of their niche.

Creatine kinase B is necessary to limit myoblast fusion during myogenesis.

Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis.

Developmental regulation of trigeminal TRPA1 by the cornea.

PURPOSE: The cornea is densely innervated with nociceptive nerves that detect deleterious stimuli at the ocular surface and transduce these stimuli as sensations of pain. Thus, nociception is a major factor involved in preventing damage to corneal tissues. One class of molecules that is thought to be involved in detecting such stimuli is the transient receptor potential (TRP) family of ion channels. However, little is known about the acquisition of these channels during corneal development. Therefore, the present study examined the developmental acquisition of these receptors and elucidated certain parameters involved in this acquisition. METHODS: Quantitative RT-PCR was used to measure the expression of genes including TRPA and Ret in vivo. In vitro cocultures between cornea and the ophthalmic lobe of the trigeminal ganglion were used to test interactions between nerves and corneas along with recombinant proteins. RESULTS: TRPA1 mRNA showed a progressive temporal increase in the ophthalmic lobe of the trigeminal ganglion in vivo during embryonic development. In vitro, TRPA1 expression was significantly increased in the ganglion when cocultured with cornea, compared to ganglia cultured alone. Similarly, the addition of exogenous neurotrophin-3 (NT3) protein to cultured ganglia increased the expression of TRPA1 more than 100-fold. Addition of NT3 and neurturin synergistically increased TRPA1 expression in embryonic day (E)8 ganglia, but this effect was lost at E12. At E8, Ret+ nonpeptidergic neurons are specified in the trigeminal ganglion. CONCLUSIONS: Corneal-derived factors increase TRPA1 expression in trigeminal nonpeptidergic neurons during their embryonic specification.

Interplay of Nkx3.2, Sox9 and Pax3 regulates chondrogenic differentiation of muscle progenitor cells.

Muscle satellite cells make up a stem cell population that is capable of differentiating into myocytes and contributing to muscle regeneration upon injury. In this work we investigate the mechanism by which these muscle progenitor cells adopt an alternative cell fate, the cartilage fate. We show that chick muscle satellite cells that normally would undergo myogenesis can be converted to express cartilage matrix proteins in vitro when cultured in chondrogenic medium containing TGFß3 or BMP2. In the meantime, the myogenic program is repressed, suggesting that muscle satellite cells have undergone chondrogenic differentiation. Furthermore, ectopic expression of the myogenic factor Pax3 prevents chondrogenesis in these cells, while chondrogenic factors Nkx3.2 and Sox9 act downstream of TGFß or BMP2 to promote this cell fate transition. We found that Nkx3.2 and Sox9 repress the activity of the Pax3 promoter and that Nkx3.2 acts as a transcriptional repressor in this process. Importantly, a reverse function mutant of Nkx3.2 blocks the ability of Sox9 to both inhibit myogenesis and induce chondrogenesis, suggesting that Nkx3.2 is required for Sox9 to promote chondrogenic differentiation in satellite cells. Finally, we found that in an in vivo mouse model of fracture healing where muscle progenitor cells were lineage-traced, Nkx3.2 and Sox9 are significantly upregulated while Pax3 is significantly downregulated in the muscle progenitor cells that give rise to chondrocytes during fracture repair. Thus our in vitro and in vivo analyses suggest that the balance of Pax3, Nkx3.2 and Sox9 may act as a molecular switch during the chondrogenic differentiation of muscle progenitor cells, which may be important for fracture healing.

Developmental regulation of the nuclear ferritoid-ferritin complex of avian corneal epithelial cells: roles of systemic factors and thyroxine.

Previously we observed that avian corneal epithelial cells protect their DNA from oxidative damage by having the iron-sequestering molecule ferritin - normally cytoplasmic - in a nuclear location. This localization involves a developmentally-regulated ferritin-like protein - ferritoid - that initially serves as the nuclear transporter, and then as a component of a ferritoid-ferritin complex that is half the size of a typical ferritin and binds to DNA. We also observed that developmentally, the synthesis of ferritin and ferritoid are regulated coordinately - with ferritin being predominantly translational and ferritoid transcriptional. In the present study we examined whether the mechanism(s) involved in this regulation reside within the cornea itself, or alternatively involve a systemic factor(s). For this, we explanted embryonic corneas of one age to the chorioallantoic membrane (CAM) of host embryos of a different age - all prior to the initiation of ferritin synthesis. Consistent with systemic regulation, the explants initiated the synthesis of both ferritin and ferritoid in concert with that of the host. We then examined whether this systemic regulation might involve thyroxine - a hormone with broad developmental effects. Employing corneal organ cultures, we observed that thyroxine initiated the synthesis of both components in a manner similar to that which occurs in vivo (i.e. ferritin was translational and ferritoid transcriptional).

Delta-like 4 induces notch signaling in macrophages: implications for inflammation.

BACKGROUND: Activated macrophages contribute to the pathogenesis of inflammatory diseases such as atherosclerosis. Although Notch signaling participates in various aspects of immunity, its role in macrophage activation remains undetermined. METHODS AND RESULTS: To explore the role of Notch signaling in inflammation, we examined the expression and activity of Notch pathway components in human primary macrophages in vitro and in atherosclerotic plaques. Macrophages in culture express various Notch pathway components including all 4 receptors (Notch1 to Notch4). Notch3 selectively increased during macrophage differentiation; however, silencing by RNA interference demonstrated that all receptors are functional. The ligand Delta-like 4 (Dll4) increased in macrophages exposed to proinflammatory stimuli such as lipopolysaccharide, interleukin-1beta, or minimally-modified low-density lipoprotein in a Toll-like receptor 4- and nuclear factor-kappaB-dependent fashion. Soluble Dll4 bound to human macrophages. Coincubation of macrophages with cells that expressed Dll4 triggered Notch proteolysis and activation; increased the transcription of proinflammatory genes such as inducible nitric oxide synthase, pentraxin 3 and Id1; resulted in activation of mitogen-activated protein kinase, Akt, and nuclear factor-kappaB pathways; and increased the expression of Dll4 in macrophages. Notch3 knockdown during macrophage differentiation decreased the transcription of genes that promote inflammation, such as inducible nitric oxide synthase, pentraxin 3, Id1, and scavenger receptor-A. These in vitro findings correlate with results of quantitative immunohistochemistry, which demonstrated the presence of Dll4 and other Notch components within macrophages in atherosclerotic plaques. CONCLUSION: Dll4-triggered Notch signaling may mediate inflammatory responses in macrophages and promote inflammation.