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Chronic low-grade adipose inflammation, characterized by aberrant adipokine production and pro-inflammatory macrophage activation/polarization is associated with increased risk of breast cancer. Adipocyte fatty acid composition is influenced by dietary availability and may regulate adipokine secretion and adipose inflammation. After feeding F344 rats for 20 weeks with a Western diet or a fish oil-supplemented diet, we cultured primary rat adipose tissue in a three-dimensional explant culture and collected the conditioned medium. The rat adipose tissue secretome was assayed using the Proteome Profiler Cytokine XL Array, and adipose tissue macrophage polarization (M1/M2 ratio) was assessed using the iNOS/ARG1 ratio. We then assessed the adipokine's effects upon stem cell self-renewal using primary human mammospheres from normal breast mammoplasty tissue. Adipose from rats fed the fish oil diet had an ω-3:ω-6 fatty acid ratio of 0.28 compared to 0.04 in Western diet rats. The adipokine profile from the fish oil-fed rats was shifted toward adipokines associated with reduced inflammation compared to the rats fed the Western diet. The M1/M2 macrophage ratio decreased by 50% in adipose of fish oil-fed rats compared to that from rats fed the Western diet. Conditioned media from rats fed the high ω-6 Western diet increased stem cell self-renewal by 62%±9% (X¯%±SD) above baseline compared to only an 11%±11% increase with the fish oil rat adipose. Modulating the adipokine secretome with dietary interventions therefore may alter stromal-epithelial signaling that plays a role in controlling mammary stem cell self-renewal., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multiple mechanisms are likely to account for the link between obesity and increased risk of postmenopausal breast cancer. Two adipokines, leptin and adiponectin, are of particular interest due to their opposing biologic functions and associations with breast cancer risk. In the current study, we investigated the effects of leptin and adiponectin on normal breast epithelial stem cells. Levels of leptin in human adipose explant-derived conditioned media positively correlated with the size of the normal breast stem cell pool. In contrast, an inverse relationship was found for adiponectin. Moreover, a strong linear relationship was observed between the leptin/adiponectin ratio in adipose conditioned media and breast stem cell self-renewal. Consistent with these findings, exogenous leptin stimulated whereas adiponectin suppressed breast stem cell self-renewal. In addition to local in-breast effects, circulating factors, including leptin and adiponectin, may contribute to the link between obesity and breast cancer. Increased levels of leptin and reduced amounts of adiponectin were found in serum from obese compared with age-matched lean postmenopausal women. Interestingly, serum from obese women increased stem cell self-renewal by 30% compared with only 7% for lean control serum. Taken together, these data suggest a plausible explanation for the obesity-driven increase in postmenopausal breast cancer risk. Leptin and adiponectin may function as both endocrine and paracrine/juxtacrine factors to modulate the size of the normal stem cell pool. Interventions that disrupt this axis and thereby normalize breast stem cell self-renewal could reduce the risk of breast cancer., (©2015 American Association for Cancer Research.)

Proper, graded communication between different cell types is essential for normal development and function. In the nervous system, heart, and for some cancer cells, part of this communication requires signaling by soluble and membrane-bound factors produced by the NRG1 gene. We have previously shown that glial-derived neurotrophic factors activate a rapid, localized release of soluble neuregulin from neuronal axons that can, in turn promote proper axoglial development (Esper, R. M., and Loeb, J. A. (2004) J. Neurosci. 24, 6218-6227). Here we elucidate the mechanism of this localized, regulated release by implicating the delta isoform of protein kinase C (PKC). Blocking the PKC delta isoform with either rottlerin, a selective antagonist, or small interference RNA blocks the regulated release of neuregulin from both transfected cells and primary neuronal cultures. PKC activation also leads to the rapid phosphorylation of the pro-NRG1 cytoplasmic tail on serine residues adjacent to the membrane-spanning segment, that, when mutated markedly reduce the rate of NRG1 activity release. These findings implicate this specific PKC isoform as an important factor for the cleavage and neurotrophin-regulated release of soluble NRG1 forms that have important effects in nervous system development and disease.

Neurons in the nascent dorsal root ganglia are born and differentiate in a complex cellular milieu composed of postmitotic neurons, and mitotically active glial and neural progenitor cells. Neurotrophic factors such as NT-3 are critically important for promoting the survival of postmitotic neurons in the DRG. However, the factors that regulate earlier events in the development of the DRG such as the mitogenesis of DRG progenitor cells and the differentiation of neurons are less defined. Here we demonstrate that both NT-3 and CNTF induce distinct dose-dependent responses on cells in the immature DRG: at low concentrations, they induce the proliferation of progenitor cells while at higher concentrations they promote neuronal differentiation. Furthermore, the mitogenic response is indirect; that is, NT-3 and CNTF first bind to nascent neurons in the DRG--which then stimulates those neurons to release mitogenic factors including neuregulin. Blockade of this endogenous neuregulin activity completely blocks the CNTF-induced proliferation and reduces about half of the NT-3-mediated proliferation. Thus, the genesis and differentiation of neurons and glia in the DRG are dependent upon reciprocal interactions among nascent neurons, glia, and mitotically active progenitor cells.

The neuregulins are a family of growth and differentiation factors with a wide range of functions in the nervous system. The power and diversity of the neuregulin signaling system comes in part from a large number of alternatively-spliced forms of the NRG1 gene that can produce both soluble and membrane-bound forms. The soluble forms of neuregulin are unique from other factors in that they have a structurally distinct heparin-binding domain that targets and potentiates its actions. In addition, a finely tuned, bidirectional mechanism regulates when and where neuregulin is released from neurons in response to neurotrophic factors produced by both neuronal targets and supporting glial cells. Together, this produces a balanced intercellular signaling system that can be localized to distinct regions for both normal development and maintenance of the mature nervous system. Recent evidence suggests that neuregulin signaling plays important roles in many neurological disorders including multiple sclerosis, traumatic brain and spinal cord injury, peripheral neuropathy, and schizophrenia. Here, we review the basic biology of neuregulins and relate this to research suggesting their involvement with and potential therapeutic uses for neurological disorders.

The signals that determine whether axons are ensheathed or myelinated by Schwann cells have long been elusive. We now report that threshold levels of neuregulin-1 (NRG1) type III on axons determine their ensheathment fate. Ensheathed axons express low levels whereas myelinated fibers express high levels of NRG1 type III. Sensory neurons from NRG1 type III deficient mice are poorly ensheathed and fail to myelinate; lentiviral-mediated expression of NRG1 type III rescues these defects. Expression also converts the normally unmyelinated axons of sympathetic neurons to myelination. Nerve fibers of mice haploinsufficient for NRG1 type III are disproportionately unmyelinated, aberrantly ensheathed, and hypomyelinated, with reduced conduction velocities. Type III is the sole NRG1 isoform retained at the axon surface and activates PI 3-kinase, which is required for Schwann cell myelination. These results indicate that levels of NRG1 type III, independent of axon diameter, provide a key instructive signal that determines the ensheathment fate of axons.

The proper function of neuromuscular junctions requires an extremely high density of acetylcholine receptors (AChRs) that may be achieved from neuron-derived factors including agrin and neuregulin. Here, we show that neuregulin-1 and agrin co-localize at neuromuscular junctions in vivo and form complexes when co-transfected into COS-7 cells. When these COS-7 cells are cultured with myotubes, synergistic effects are observed for AChR clustering, membrane insertion of new AChRs, and induction of AChR mRNA. Even a muscle form of agrin that lacks intrinsic clustering activities by itself, significantly enhances neuregulin-induced clustering and insertion of AChRs. While the heparin-binding (A) domain of agrin is required for agrin localization in the extracellular matrix adjacent to AChR clusters, the heparan sulfate-containing domain of agrin is needed for the synergistic effects and co-localization with neuregulin-1. These results suggest that matrix interactions between exogenously supplied agrin and neuregulin-1 on the muscle surface provide a localized source of signaling factors needed to produce high densities of AChRs at neuromuscular junctions.

During peripheral nervous system development, Schwann cells are precisely matched to the axons that they support. This is mediated by axonal neuregulins that are essential for Schwann cell survival and differentiation. Here, we show that sensory and motor axons rapidly release heparin-binding forms of neuregulin in response to Schwann cell-derived neurotrophic factors in a dose-dependent manner. Neuregulin release occurs within minutes, is saturable, and occurs from axons that were isolated using a newly designed chamber slide apparatus. Although NGF and glial cell line-derived neurotrophic factor (GDNF) were the most potent neurotrophic factors to release neuregulin from sensory neurons, GDNF and BDNF were most potent for motor neurons and were the predominant neuregulin-releasing neurotrophic factors produced by cultured Schwann cells. Comparable levels of neuregulin could be released at a similar rate from neurons after protein kinase C activation with the phorbol ester, phorbol 12-myristate 13-acetate, which has also been shown to promote the cleavage and release of neuregulin from its transmembrane precursor. The rapid release of neuregulin from axons in response to Schwann cell-derived neurotrophic factors may be part of a spatially restricted system of communication at the axoglial interface important for proper peripheral nerve development, function, and repair.

Exposure to environmental heavy metals has been reported to affect the immune system. Here, we tested the hypothesis that Hg(+2), acting through membrane proteins, disrupts metabolic dynamics and downstream cell functions in human neutrophils. We found that HgCl(2) inhibited: (1) polarization and (2) immunoglobulin (Ig)G-mediated phagocytosis of sheep erythrocytes in a dose-dependent manner from 2.5 to 10 microM. Because these activities have been linked with pro-inflammatory signalling, we also studied the effects of HgCl(2) on intracellular signalling by measuring protein tyrosine phosphorylation. HgCl(2) at doses = 1 microM increased tyrosine phosphorylation. We also studied the effect of HgCl(2) on neutrophil metabolism by measuring NAD(P)H autofluorescence as an indicator of intracellular NAD(P)H concentration. After HgCl(2) treatment, we found that normal sinusoidal NAD(P)H oscillations became incoherent. We recently reported that the NAD(P)H oscillation frequency is affected by cell migration and activation, which can in turn be regulated by integrin-mediated signalling. Therefore, we examined the effects of HgCl(2) on cell surface distribution of membrane proteins. After exposure to environmentally relevant concentrations of HgCl(2) we found that CR3, but not other membrane proteins (e.g. uPAR, Fc gamma RIIA and the formyl peptide receptor), became clustered on cell surfaces. We suggest that HgCl2 disrupts integrin signalling/functional pathways in neutrophils.