Loss of EMP2 Inhibits Melanogenesis of MNT1 Melanoma Cells via Regulation of TRP-2

Melanogenesis is the production of melanin from tyrosine by a series of enzyme-catalyzed reactions, in which tyrosinase and DOPA oxidase play key roles. The melanin content in the skin determines skin pigmentation. Abnormalities in skin pigmentation lead to various skin pigmentation disorders. Recent research has shown that the expression of EMP2 is much lower in melanoma than in normal melanocytes, but its role in melanogenesis has not yet been elucidated. Therefore, we investigated the role of EMP2 in the melanogenesis of MNT1 human melanoma cells. We examined TRP-1, TRP-2, and TYR expression levels during melanogenesis in MNT1 melanoma cells by gene silencing of EMP2. Western blot and RT-PCR results confirmed that the expression levels of TYR and TRP-2 were decreased when EMP2 expression was knocked down by EMP2 siRNA in MNT1 cells, and these changes were reversed when EMP2 was overexpressed. We verified the EMP2 gene was knocked out of the cell line (EMP2 CRISPR/Cas9) by using a CRISPR/Cas9 system and found that the expression levels of TRP-2 and TYR were significantly lower in the EMP2 CRISPR/Cas9 cell lines. Loss of EMP2 also reduced migration and invasion of MNT1 melanoma cells. In addition, the melanosome transfer from the melanocytes to keratinocytes in the EMP2 KO cells cocultured with keratinocytes was reduced compared to the cells in the control coculture group. In conclusion, these results suggest that EMP2 is involved in melanogenesis via the regulation of TRP-2 expression.     

Bone morphogenetic protein regulation of enteric neuronal phenotypic diversity: relationship to timing of cell cycle exit

The effects of bone morphogenetic protein (BMP) signaling on enteric neuron development were examined in transgenic mice overexpressing either the BMP inhibitor, noggin, or BMP4 under control of the neuron specific enolase (NSE) promoter. Noggin antagonism of BMP signaling increased total numbers of enteric neurons and those of subpopulations derived from precursors that exit the cell cycle early in neurogenesis (serotonin, calretinin, calbindin). In contrast, noggin overexpression decreased numbers of neurons derived from precursors that exit the cell cycle late (gamma-aminobutyric acid, tyrosine hydroxylase [TH], dopamine transporter, calcitonin gene-related peptide, TrkC). The numbers of TH- and TrkC-expressing neurons were increased by overexpression of BMP4. These observations are consistent with the idea that phenotypic expression in the enteric nervous system (ENS) is determined, in part, by the number of proliferative divisions neuronal precursors undergo before their terminal mitosis. BMP signaling may thus regulate enteric neuronal phenotypic diversity by promoting the exit of precursors from the cell cycle. BMP2 increased the numbers of TH- and TrkC-expressing neurons developing in vitro from immunoselected enteric crest-derived precursors; BMP signaling may thus also specify or promote the development of dopaminergic TrkC/NT-3-dependent neurons. The developmental defects in the ENS of noggin-overexpressing mice caused a relatively mild disturbance of motility (irregular rapid transit and increased stool frequency, weight, and water content). Although the function of the gut thus displays a remarkable tolerance for ENS defects, subtle functional abnormalities in motility or secretion may arise when ENS defects short of aganglionosis occur during development.     

Multiple modes of amplification of synaptic inhibition to motoneurons by persistent inward currents

The ability of inhibitory synaptic inputs to dampen the excitability of motoneurons is augmented when persistent inward currents (PICs) are activated. This amplification could be due to an increase in the driving potential of inhibitory synapses or the deactivation of the channels underlying PICs. Our goal was to determine which mechanism leads to the amplification of inhibitory inputs by PICs. To reach this goal, we measured inhibitory postsynaptic currents (IPSCs) in decerebrate cats during somatic voltage-clamp steps. These IPSCs were generated by tonic activation of Renshaw cells. The IPSCs exhibited a rapid rise and a slower decay to a plateau level. Activation of PICs always led to an increase in the peak of the IPSC, but the amount of decay after the peak of the IPSC was inversely related to the size of the IPSC. These results were replicated in simulations based on compartmental models of motoneurons incorporating distributions of Renshaw cell synapses based on anatomical observations, and L-type calcium channels distributed as 100-microm-long hot spots centered 100 to 400 microm away from the soma. For smaller IPSCs, amplification by PICs was due to an increase in the driving force of the inhibitory synaptic current. For larger IPSCs, amplification was caused by deactivation of the channels underlying PICs leading to a lesser decay of the IPSCs. As a result of this change in the time course of the IPSC, deactivation of the channels underlying PICs leads to a greater amplification of the total inhibitory synaptic current.     

Growth/differentiation factor 5 enhances chondrocyte maturation.

Growth/differentiation factor 5 (GDF5) is required for limb mesenchymal cell condensation and joint formation during skeletogenesis. Here, we use a model consisting of long-term, high-density cultures of chick embryonic limb mesenchymal cells, which undergo the entire life history of chondrocyte development, to examine the effects of GDF5 overexpression on chondrocyte maturation. Exposure to GDF5 significantly enhanced chondrocyte hypertrophy and maturation, as determined by the presence of alkaline phosphatase activity, collagen type X protein production, and the presence of a sulfated proteoglycan-rich extracellular matrix. Histologic analysis also revealed an increase in cell volume and cellular encasement in larger lacunae in GDF5-treated cultures. Taken together, these results support a role for GDF5 in influencing chondrocyte maturation and the induction of hypertrophy in the late stages of embryonic cartilage development, and provide additional mechanistic insights into the role of GDF5 in skeletal development.     

Glucocorticoids promote chondrogenic differentiation of adult human mesenchymal stem cells by enhancing expression of cartilage extracellular matrix genes

In the adult human, mesenchymal stem cells (hMSCs) resident in the bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. Glucocorticoids (GCs) are required for chondrogenic differentiation of hMSCs in vitro; however, the exact role of GCs in this process is not known. In this study, we examined the effects of dexamethasone (DEX) on chondrogenic differentiation of hMSCs in the presence or absence of DEX, transforming growth factor-beta (TGF-beta), or DEX plus TGF-beta. GC treatment upregulated gene expression of cartilage matrix components aggrecan, dermatopontin, and collagen type XI; enhanced TGF-beta-mediated upregulation of collagen type II and cartilage oligomeric matrix protein; and increased aggrecan and collagen type II production as well as cartilage matrix-sulfated proteoglycans as assessed by immunohistochemistry and alcian blue staining. Inclusion of an antagonist of GCs inhibited expression of chondrogenic differentiation markers, suggesting that the GC effects during chondrogenesis are mediated by the GC receptor (GR). Steady levels of the major active form of GR, GRalpha, were detected in both undifferentiated and differentiating hMSCs, whereas the dominant-negative isoform GRbeta, present at low levels in undifferentiated hMSCs, was downregulated during chondrogenesis. In the presence of DEX and TGF-beta, expression of a collagen type II gene promoter luciferase reporter construct in hMSCs was upregulated. However, coexpression of GRbeta dramatically inhibited promoter activity, suggesting that GRalpha is required for GC-mediated modulation of chondrogenesis and that GCs may play an important role in the maintenance of cartilage homeostasis.     

Computational estimation of the distribution of L-type Ca(2+) channels in motoneurons based on variable threshold of activation of persistent inward currents

In the presence of neuromodulators such as serotonin and noradrenaline, motoneurons exhibit persistent inward currents (PICs) that serve to amplify synaptic inputs. A major component of these PICs is mediated by L-type Ca(2+) channels. Estimates based on electrophysiological studies indicate that these channels are located on the dendrites, but immunohistochemical studies of their precise distribution have yielded different results. Our goal was to determine the distribution of these channels using computational methods. A theoretical analysis of the activation of PICs by a somatic current injection in the absence or presence of synaptic activity suggests that L-type Ca(2+) channels may be segregated to discrete hot spots 25-200 microm long and centered 100-400 microm from the soma in the dendritic tree. Compartmental models based on detailed anatomical measurements of the structure of feline neck motoneurons with L-type Ca(2+) channels incorporated in these regions produced plateau potentials resulting from PIC activation. Furthermore, we replicated the experimental observation that the somatic threshold at which PICs were activated was depolarized by tonic activation of inhibitory synapses and hyperpolarized by tonic activation of excitatory synapses. Models with L-type Ca(2+) channels distributed uniformly were unable to replicate the change in somatic threshold of PIC activation. Therefore we conclude that the set of L-type Ca(2+) channels mediating plateau potentials is restricted to discrete regions in the dendritic tree. Furthermore, this distribution leads to the compartmentalization of the dendritic tree of motoneurons into subunits whose sequential activation lead to the graded amplification of synaptic inputs.     

Fabrication and characterization of six electrospun poly(alpha-hydroxy ester)-based fibrous scaffolds for tissue engineering applications

The most common synthetic biodegradable polymers being investigated for tissue engineering applications are FDA approved, clinically used poly(alpha-hydroxy esters). To better assess the applicability of the electrospinning technology for scaffold fabrication, six commonly used poly(alpha-hydroxy esters) were used to prepare electrospun fibrous scaffolds, and their physical and biological properties were also characterized. Our results suggest that specific, optimized fabrication parameters are required for each polymer to produce scaffolds that consist of uniform structures morphologically similar to native extracellular matrix. Scanning electron microscopy (SEM) revealed a highly porous, three-dimensional structure for all scaffolds, with average fiber diameter ranging from 300nm to 1.5microm, depending on the polymer type used. The poly(glycolic acid) (PGA) and poly(d,l-lactic-co-glycolic acid 50:50) (PLGA5050) fibrous structures were mechanically stiffest, whereas the poly(l-lactic acid) (PLLA) and poly(epsilon-caprolactone) (PCL) scaffolds were most compliant. Upon incubation in physiological solution, severe structural destruction due to polymer degradation was found in the PGA, poly(d,l-lactic acid) (PDLLA), PLGA5050, and poly(d,l-lactic-co-glycolic acid 85:15) (PLGA8515) fibrous scaffolds, whereas PLLA and PCL fibrous scaffolds maintained a robust scaffold structure during the same time period, based on macroscopic and SEM observations. In addition, PLLA scaffolds supported the highest rate of proliferation of seeded cells (chondrocytes and mesenchymal stem cells) than other polymeric scaffolds. Our findings showed that PLLA and PCL based fibrous scaffolds exhibited the most optimal structural integrity and supported desirable cellular response in culture, suggesting that such scaffolds may be promising candidate biomaterials for tissue engineering applications.     

Adenoviral overexpression and small interfering RNA suppression demonstrate that plasminogen activator inhibitor-1 produces elevated collagen accumulation in normal and keloid fibroblasts

Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. We provide new evidence that both overexpression of plasminogen activator inhibitor-1 (PAI-1) and elevated collagen accumulation are intrinsic features of keloid fibroblasts and that these characteristics are causally linked. Using seven strains each of early passage normal and keloid fibroblasts, the keloid strains exhibited inherently elevated collagen accumulation and PAI-1 expression in serum-free, 0.1% ITS+ culture; larger increases in these parameters occurred when cells were cultured in 3% serum. To demonstrate a causal relationship between PAI-1 overexpression and collagen accumulation, normal fibroblasts were infected with PAI-1-expressing adenovirus. Such cells exhibited a two- to fourfold increase in the accumulation of newly synthesized collagen in a viral dose-dependent fashion in both monolayers and fibrin gel, provisional matrix-like cultures. Three different PAI-1-targeted small interfering RNAs, alone or in combination, produced greater than an 80% PAI-1 knockdown and reduced collagen accumulation in PAI-1-overexpressing normal or keloid fibroblasts. A vitronectin-binding mutant of PAI-1 was equipotent with wild-type PAI-1 in inducing collagen accumulation, whereas a complete protease inhibitor mutant retained approximately 50% activity. Thus, PAI-1 may use more than its protease inhibitory activity to control keloid collagen accumulation. PAI-1-targeted interventions, such as small interfering RNA and lentiviral short hairpin RNA-containing microRNA sequence suppression reported here, may have therapeutic utility in the prevention of keloid scarring.