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Collective migration depends on cell-cell interactions between neighbors that contribute to their overall directionality, yet the mechanisms that control the coordinated migration of neurons remains to be elucidated. During hindbrain development, facial branchiomotor neurons (FBMNs) undergo a stereotypic tangential caudal migration from their place of birth in rhombomere (r)4 to their final location in r6/7. FBMNs engage in collective cell migration that depends on neuron-to-neuron interactions to facilitate caudal directionality. Here, we demonstrate that Cadherin-2-mediated neuron-to-neuron adhesion is necessary for directional and collective migration of FBMNs. We generated stable transgenic zebrafish expressing dominant-negative Cadherin-2 (Cdh2ΔEC) driven by the islet1 promoter. Cell-autonomous inactivation of Cadherin-2 function led to non-directional migration of FBMNs and a defect in caudal tangential migration. Additionally, mosaic analysis revealed that Cdh2ΔEC-expressing FBMNs are not influenced to migrate caudally by neighboring wild-type FBMNs due to a defect in collective cell migration. Taken together, our data suggest that Cadherin-2 plays an essential cell-autonomous role in mediating the collective migration of FBMNs.

Exposure of the lung to ionizing radiation, such as during radiotherapy, can result in pulmonary fibrosis (PF), which has few treatment options. PF is characterized by an accumulation of extracellular matrix proteins that form scar tissue, resulting in dyspnea, disruption of gas exchange, and even death. We and others have shown that metabolic reprogramming is a hallmark of idiopathic pulmonary fibrosis (IPF). IPF lung tissue, and lung fibroblasts treated with TGF-β, exhibit increased aerobic glycolysis with increased expression of lactate dehydrogenase A (LDHA) and excess production of lactate, leading to reduced extracellular pH that activates latent TGF-β. Here, we hypothesized that ionizing radiation would cause aerobic glycolytic metabolic dysregulation in primary human lung fibroblasts.Methods: Primary human lung fibroblasts (HLFs) from two non-fibrotic donors were seeded and subjected to either no treatment, TGF-β treatment (500 pg/mL), or radiation (3, 5, and 7 Gy). Cell lysates were harvested 2 and 5 days after irradiation for RNA and protein, respectively. Gene and protein expression of metabolic markers were determined by RT-PCR and western blot. TBP (RT-PCR) and GAPDH (western blots) were used as loading controls. Cell viability was estimated immediately prior to cell lysate harvest using Presto Blue.Results: Primary non-fibrotic HLFs exposed to irradiation exhibited significant upregulation of Pyruvate Dehydrogenase Kinase (PDK)1 (0.5 -3-fold, p<0.05), LDHA (1.4-fold, p<0.05), and LDHB (2-fold, p<0.05). The transcription factor FOXO1 exhibited a trend toward increased expression. Cell viability was unaffected by increased radiation dose.Conclusions: Radiation increased fibroblast expression of genes involved in aerobic glycolysis (PDK1, LDHA, LDHB), in a similar pattern to that seen in IPF fibroblasts. FOXO1, which regulates PDK1 and other genes in the glycolytic pathway, was not significantly upregulated. Radiation may alter its activity rather than mRNA levels. The metabolic changes are closely associated with creating a profibrotic extracellular environment in IPF by promoting an acidic environment. This phenomenon in fibrotic fibroblasts is similar to observations of the Warburg effect in cancer cells, where aerobic glycolysis occurs despite the presence of oxygen, allowing growth advantages. Our evidence suggests this phenomenon can be driven by radiation in lung fibroblasts and affirm that glycolytic reprogramming may also be a hallmark of radiation-induced fibrosis. Further understanding of the common mechanisms that create this metabolic shift could provide novel therapeutics for fibrosis treatment.

Pulmonary fibrosis results from excessive deposition of extracellular matrix proteins and stiffening of the interstitial matrix leading to irreversible loss of lung function. Tissue stiffness is also a pathogenic driver of fibrosis creating a pro-fibrotic feed forward loop. We have published novel data describing that IPF patients have abnormally high levels of lactate in their lungs, and that TGFβ drives fibroblasts to produce lactic acid (lactate) in culture via the enzyme lactate dehydrogenase-A (LDHA). Lactate in turn, lowers pH levels, which activates latent TGFβ, resulting in another pro-fibrotic feed-forward loop that drives fibrosis. The importance of metabolic dysregulation and tissue stiffness in pulmonary fibrosis have previously been studied in isolation, but the potential links between these two key pathways have not previously been investigated. We propose that biomechanical stress drives normal lung fibroblasts toward a pro-fibrotic phenotype in part by driving metabolic changes that result in excess production of lactate and activation of TGFβ. In this study we investigate the effect of matrix stiffness on metabolic dysregulation of lung fibroblasts when seeded on physiologically healthy (∼ 2 kPa) and fibrotic (∼ 30 kPa) substrates. We hypothesize that altered tissue stiffness enhances dysregulation of lactate metabolism and contributes to myofibroblast differentiation and pulmonary fibrosis. Methods: Primary human lung fibroblasts from non-fibrotic donors were seeded on collagen-coated polyacrylamide gels of 2-kPa and 30-kPa stiffness. Cell lysates were harvested after 48 hours after seeding on substrate. Gene expression of EEFa1a and metabolic markers were determined by RT-PCR; normalized to EEF and the 2-kPa group. Results: Primary non-fibrotic human lung fibroblasts seeded on stiff (30-kPa) substrates demonstrated upregulated glycolytic metabolism markers. The gene encoding for lactate, LDHA (3.4-fold, p=0.0041), and lactate transporters, MCT1 (3.5-fold, p=0.01) and MCT4 (3.3-fold, p=0.03), are significantly upregulated. Furthermore, the transcription factor FOXO1 (5.1-fold, p=0.2) and its target PDK1 (4.0-fold, p=0.03) are upregulated. Conclusions: Identifying pro-fibrotic mechanisms that contribute to the progression of IPF is critical to therapeutic development. We demonstrate that fibroblasts grown on matrices of stiffness equivalent to fibrotic lung tissue express not only myofibroblast differentiation markers, but also exhibit dysregulated lactate metabolism gene changes. This level of cross-talk between pro-fibrotic signals represents a novel regulatory axis and potential therapeutic target in IPF. This identifies a previously unknown link between the lactate and mechanical strain pathways and suggests that these pathways have redundancies that may limit the efficacy of therapies that target a single pathway.

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