Regulation of proximal-distal intercalation during limb regeneration in the axolotl (Ambystoma mexicanum)

Intercalation is the process whereby cells located at the boundary of a wound interact to stimulate proliferation and the restoration of the structures between the boundaries that were lost during wounding. Thus, intercalation is widely considered to be the mechanism of regeneration. When a salamander limb is amputated, the entire cascade of regeneration events is activated, and the missing limb segments and their boundaries (joints) as well as the structures within each segment are regenerated. Therefore, in an amputated limb it is not possible to distinguish between intersegmental regeneration (formation of new segments/joints) and intrasegmental regeneration (formation of structures within a given segment), and it is not possible to study the differential regulation of these two processes. We have used two models for regeneration that allow us to study these two processes independently, and report that inter- and intrasegmental regeneration are different processes regulated by different signaling pathways. New limb segments/joints can be regenerated from cells that dedifferentiate to form blastema cells in response to signaling that is mediated in part by fibroblast growth factor.     

Nerve–blastema interactions induce fibroblast growth factor-1 release during limb regeneration in Pleurodeles waltl

Previous studies have shown that both fibroblast growth factor (FGF)-1 and nerves play an important function during limb regeneration, but no correlation between these two regeneration factors has yet been demonstrated. In the present study we first establish that exogenous FGF-2, a member of the FGF family that binds to the same high-affinity receptors as FGF-1, is able to stimulate both [³H]-thymidine incorporation and the mitotic index in the mesenchyme and the epidermal cells of denervated blastemas. We then use cocultures of spinal cord and blastema on heparin-coated dishes, an in vitro system mimicking the in vivo interactions during limb regeneration, to show that interactions between nerve fibers from the spinal cord and the blastema enhance the release of bioactive FGF-1. Release of this growth factor seemed to correlate with nerve fiber regeneration, as it decreased in the presence of the dipeptide Leu-Ala, known to inhibit neurite outgrowth, while the inverse dipeptide Ala-Leu was inactive. Therefore, these results support our hypothesis that the interaction between nervous tissue and blastema is permissive for the release of FGF-1, which in turn stimulates blastema cell proliferation.     

LINE‐1 hypomethylation induced by reactive oxygen species is mediated via depletion of S‐adenosylmethionine

Whether long interspersed nuclear element‐1 (LINE‐1) hypomethylation induced by reactive oxygen species (ROS) was mediated through the depletion of S‐adenosylmethionine (SAM) was investigated. Bladder cancer (UM‐UC‐3 and TCCSUP) and human kidney (HK‐2) cell lines were exposed to 20 μM H₂O₂ for 72 h to induce oxidative stress. Level of LINE‐1 methylation, SAM and homocysteine (Hcy) was measured in the H₂O₂‐exposed cells. Effects of α‐tocopheryl acetate (TA), N‐acetylcysteine (NAC), methionine, SAM and folic acid on oxidative stress and LINE‐1 methylation in the H₂O₂‐treated cells were explored. Viabilities of cells treated with H₂O₂ were not significantly changed. Intracellular ROS production and protein carbonyl content were significantly increased, but LINE‐1 methylation was significantly decreased in the H₂O₂‐treated cells. LINE‐1 methylation was restored by TA, NAC, methionine, SAM and folic acid. SAM level in H₂O₂‐treated cells was significantly decreased, while total glutathione was significantly increased. SAM level in H₂O₂‐treated cells was restored by NAC, methionine, SAM and folic acid; while, total glutathione level was normalized by TA and NAC. Hcy was significantly decreased in the H₂O₂‐treated cells and subsequently restored by NAC. In conclusion, in bladder cancer and normal kidney cells exposed to H₂O₂, SAM and Hcy were decreased, but total glutathione was increased. Treatments with antioxidants (TA and NAC) and one‐carbon metabolites (SAM, methionine and folic acid) restored these changes. This pioneer finding suggests that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy, which consequently causes SAM depletion and eventual hypomethylation of LINE‐1. Copyright © 2015 John Wiley & Sons, Ltd.     

The role of TGF‐β1 during skeletal muscle regeneration

The injury of adult skeletal muscle initiates series of well‐coordinated events that lead to the efficient repair of the damaged tissue. Any disturbances during muscle myolysis or reconstruction may result in the unsuccessful regeneration, characterised by strong inflammatory response and formation of connective tissue, that is, fibrosis. The switch between proper regeneration of skeletal muscle and development of fibrosis is controlled by various factors. Amongst them are those belonging to the transforming growth factor β family. One of the TGF‐β family members is TGF‐β1, a multifunctional cytokine involved in the regulation of muscle repair via satellite cells activation, connective tissue formation, as well as regulation of the immune response intensity. Here, we present the role of TGF‐β1 in myogenic differentiation and muscle repair. The understanding of the mechanisms controlling these processes can contribute to the better understanding of skeletal muscle atrophy and diseases which consequence is fibrosis disrupting muscle function.     

Protease‐activated receptor‐1 modulates hippocampal memory formation and synaptic plasticity

Protease‐activated receptor‐1 (PAR1) is an unusual G‐protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1?/? mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1?/? mice have deficits in hippocampus‐dependent memory. We also show that while PAR1?/? mice have normal baseline synaptic transmission at Schaffer collateral‐CA1 synapses, they exhibit severe deficits in N‐methyl‐d ‐aspartate receptor (NMDAR)‐dependent long‐term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR‐mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR‐dependent processes subserving memory formation and synaptic plasticity.     

Msx1‐2 immunolocalization in the regenerating tail of a lizard but not in the scarring limb suggests its involvement in the process of regeneration

The immunolocalization of the muscle segmental homoeobox protein Msx1‐2 of 27–34 kDa in the regenerating tail blastema of a lizard shows prevalent localization in the apical ependyma of the regenerating spinal cord and less intense labelling in the wound epidermis, in the apical epidermal peg (AEP), and in the regenerating segmental muscles. The AEP is a micro‐region of the regenerating epidermis located at the tail tip of the blastema, likely corresponding to the AEC of the amphibian blastema. No immunolabelling is present in the wound epidermis and scarring blastema of the limb at 18–21 days of regeneration, except for sparse repairing muscles. The presence of a proximal–distal gradient of Msx1‐2 protein, generated from the apical ependyma, is suggested by the intensity of immunolabelling. The AEP and the ependyma are believed to induce and maintain tail regeneration, and this study suggests that Msx1‐2 proteins are components of the signalling system that maintains active growth of the tail blastema. The lack of activation and production of Msx1‐2 protein in the limb are likely due to the intense inflammatory reaction following amputation. This study confirms that, like during regeneration in fishes and amphibians, also the blastema of lizards utilizes common signalling pathways for maintaining regeneration.     

Cementum protein 1‐derived peptide (CEMP 1‐p1) modulates hydroxyapatite crystal formation in vitro

A cementum protein 1‐derived peptide (CEMP1‐p1) consisting of 20 amino acids from the CEMP1's N‐terminus region: MGTSSTDSQQAGHRRCSTSN, and its role on the mineralization process in a cell‐free system, was characterized. CEMP1‐p1's physicochemical properties, crystal formation, and hydroxyapatite (HA) nucleation assays were performed. Crystals induced by CEMP1‐p1 were analyzed by scanning electron microscopy, Fourier‐transform infrared spectroscopy‐attenuated total reflectance (FTIR‐ATR), X‐ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM), and atomic force microscopy. The results indicate that CEMP1‐p1 lacks secondary structure, forms nanospheres that organize into three‐dimensional structures, possesses affinity to HA, and induces its nucleation. CEMP1‐p1 promotes the formation of spherical structures composed by densely packed prism‐like crystals, which revealed a Ca/P ratio of 1.56, corresponding to HA. FTIR‐ATR showed predominant spectrum peaks that correspond and are characteristic of HA and octacalcium phosphate (OCP). Analysis by XRD indicates that the crystals show planes with a preferential crystalline orientation for HA and for OCP. HRTEM showed interplanar distances that correspond to crystalline planes of HA and OCP. Crystals are composed by superimposed lamellae, which exhibit epitaxial growth, and each layer of the crystals is structured by nanocrystals. This study reveals that CEMP1‐p1 regulates HA crystal formation, somehow mimicking the in vivo process of mineralized tissues bioformation.     

Protease‐activated receptor‐1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy

End‐stage renal disease, the final stage of all chronic kidney disorders, is associated with renal fibrosis and inevitably leads to renal failure and death. Transition of tubular epithelial cells (TECs) into mesenchymal fibroblasts constitutes a proposed mechanism underlying the progression of renal fibrosis and here we assessed whether protease‐activated receptor (PAR)‐1, which recently emerged as an inducer of epithelial‐to‐mesenchymal transition (EMT), aggravates renal fibrosis. We show that PAR‐1 activation on TECs reduces the expression of epithelial markers and simultaneously induces mesenchymal marker expression reminiscent of EMT. We next show that kidney damage was reduced in PAR‐1‐deficient mice during unilateral ureter obstruction (UUO) and that PAR‐1‐deficient mice develop a diminished fibrotic response. Importantly, however, we did hardly observe any signs of mesenchymal transition in both wild‐type and PAR‐1‐deficient mice suggesting that diminished fibrosis in PAR‐1‐deficient mice is not due to reduced EMT. Instead, the accumulation of macrophages and fibroblasts was significantly reduced in PAR‐1‐deficient animals which were accompanied by diminished production of MCP‐1 and TGF‐β. Overall, we thus show that PAR‐1 drives EMT of TECs in vitro and aggravates UUO‐induced renal fibrosis although this is likely due to PAR‐1‐dependent pro‐fibrotic cytokine production rather than EMT.     

Single‐nucleotide polymorphism (A118G) in exon 1 of OPRM1 gene causes alteration in downstream signaling by mu‐opioid receptor and may contribute to the genetic risk for addiction

The opioid receptor mu1 (OPRM1) mediates the action of morphine. Although genetic background plays an important role in the susceptibility toward abuse of drugs as evident from familial, adoption and twin studies, association of specific single‐nucleotide polymorphisms of OPRM1 gene with narcotic addiction is to be established. Here, we demonstrate the involvement of A118G polymorphism of exon1 of human OPRM1 gene (hOPRM1), with heroin and alcohol addiction, in a population in eastern India. Statistical analysis exhibited a significant association of G allele with both heroin and alcohol addiction with a risk factor of P_trend < 0.05. The functional significance of G allele in A118G single‐nucleotide polymorphisms was evaluated by studying the regulation of protein kinase A (PKA), pCREB, and pERK1/2 by morphine in Neuro 2A cells, stably transfected with either wild type or A118G mutant hOPRM1. Unlike acute morphine treatment, both chronic morphine exposure and withdrawal precipitated by naloxone were differentially regulated by A118 and G118 receptor isoforms when both PKA and pERK1/2 activities were compared. Results suggest that the association of A118G polymorphism to heroin and alcohol addiction may be because of the altered regulation of PKA and pERK1/2 during opioid and alcohol exposures.     

Msx1 expressing mesoderm is important for the apical ectodermal ridge (AER)-signal transfer in chick limb development

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1 , in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER-mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero-proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re-expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf-8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.