Seok Jong Hong

Orphan nuclear receptor Nurr1 directly transactivates the promoter activity of the tyrosine hydroxylase gene in a cell-specific manner

Tyrosine hydroxylase (TH) catalyzes the first and rate‐limiting step of catecholamine synthesis and its expression is necessary for neurotransmitter specification of all catecholaminergic neurons, while dopamine β‐hydroxylase (DBH) is essential for the noradrenergic phenotype. In the present study, we show that Nurr1, an orphan nuclear receptor critical for dopaminergic (DA) neuron development, directly transactivates the promoter activity of the TH gene in a cell type‐dependent manner, while it does not regulate the DBH promoter. Consistent with these results, only the TH promoter contains multiple sequence motifs homologous to the known Nurr1‐binding motif, NBRE. TH promoter deletional analysis indicates that < 1.0 kb upstream sequences, encompassing three NBRE‐like motifs (i.e. NL1, NL2 and NL3) are mostly responsible for the effects of Nurr1. Among these potential motifs, site‐directed mutational analysis showed that NL1, residing from − 35 to − 28 bp, was most critical for mediating the transactivation by Nurr1. Strikingly, however, both DNase I footprinting and electrophoretic mobility shift assays showed that NL3, but not NL1 or NL2, has high binding affinity to Nurr1. To determine whether the proximity of these motifs may be important for transactivation by Nurr1 in the transient transfection assay, we generated reporter gene constructs in which NL3 is immediately proximal to the TATA box. Indeed, NL3 was more efficient in this position than NL1 or NL2 for mediating the transactivation by Nurr1. Our results suggest that Nurr1 may play a direct role for specification of DA neurotransmitter identity by activating TH gene transcription in a cell context‐dependent manner.

Development of a novel, highly quantitative in vivo model for the study of biofilm-impaired cutaneous wound healing

A growing body of evidence suggests that in addition to hypoxia, ischemia‐reperfusion injury, and intrinsic host factors, bacterial biofilms represent a fourth major pillar in chronic wound pathogenesis. Given that most studies to date rely on in vitro or observational clinical data, our aim was to develop a novel, quantitative animal model enabling further investigation of the biofilm hypothesis in vivo. Dermal punch wounds were created in New Zealand rabbit ears, and used as uninfected controls, or inoculated with green fluorescent protein‐labeled Staphylococcus aureus to form wounds with bacteria predominantly in the planktonic or biofilm phase. Epifluorescence and scanning electron microscopy revealed that S. aureus rapidly forms mature biofilm in wounds within 24 hours of inoculation, with persistence of biofilm viability over time seen through serial bacterial count measurement and laser scanning confocal imaging at different time points postwounding and inoculation. Inflammatory markers confirmed that the biofilm phenotype creates a characteristic, sustained, low‐grade inflammatory response, and that over time biofilm impairs epithelial migration and granulation tissue in‐growth, as shown histologically. We have established and validated a highly quantitative, reproducible in vivo biofilm model, while providing evidence that the biofilm phenotype specifically contributes to profound cutaneous wound healing impairment. Our model highlights the importance of bacterial biofilms in chronic wound pathogenesis, providing an in vivo platform for further inquiry into the basic biology of bacterial biofilm–host interaction and high‐throughput testing of antibiofilm therapeutics.

Regulation of the tyrosine hydroxylase and dopamine β-hydroxylase genes by the transcription factor AP-2

The retinoic acid‐inducible and developmentally regulated transcription factor AP‐2 plays an important role during development. In adult mammals, AP‐2 is expressed in both neural and non‐neural tissues. However, the function of AP‐2 in different neuronal phenotypes is poorly understood. In this study, transcriptional regulation of tyrosine hydroxylase (TH) and dopamine β‐hydroxylase (DBH) genes by AP‐2 was investigated. AP‐2 binding sites were identified in the upstream regions of both genes. Electrophoretic mobility shift assays (EMSA) and DNase I footprinting analyses indicate that the AP−2 interaction with these motifs is more prominent in catecholaminergic SK‐N‐BE(2)C and CATH.a than in non‐catecholaminergic HeLa and HepG2 cell lines. Exogenous expression of AP‐2 robustly transactivated TH and DBH promoter activities in non‐catecholaminergic cell lines. While AP‐2 regulates the DBH promoter activity via a single site, transactivation of the TH promoter by AP‐2 appears to require multiple sites. In support of this, mutation of multiple AP‐2 binding sites but not that of single site diminished the basal promoter activity of the TH gene in cell lines that express TH and abolished transactivation by exogenous AP‐2 expression in cell lines that do not express TH. In contrast, mutation of a single AP‐2 binding site of the DBH gene completely abolished transactivation by AP‐2. Double‐label immunohistochemistry showed that AP‐2 is coexpressed with TH in noradrenergic and adrenergic neurons in both the central and peripheral nervous systems of adult rodents. Numerous non‐catecholaminergic cell groups within the spinal cord, medulla, cerebellum, and pons also express AP‐2. The concentration of AP‐2 in dorsomedial locations along the neuraxis suggests a regionally specific role for this transcription factor in the regulation of neuronal function. Based on these findings we propose that AP‐2 may coregulate TH and DBH gene expression and thus participate in expression/maintenance of neurotransmitter phenotypes in (nor)adrenergic neurons and neuroendocrine cells.

Regulation of tyrosine hydroxylase gene transcription by the cAMP-signaling pathway: Involvement of multiple transcription factors

The conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine by tyrosine hydroxylase (TH) is the first and rate-limiting step in biosynthesis of catecholamine neurotransmitters. TH gene expression is regulated in a cell type-specific and cAMP-dependent manner. Evidence from this laboratory and others indicates that the cAMP response element (CRE), residing at -45 to -38 bp upstream of the transcription initiation site, is essential for both basal and cAMP-inducible transcription of the TH gene. To understand the control mechanisms of TH gene transcription in greater detail, we sought to identify and characterize the transcription factors involved in recognition and activation of the CRE of the TH gene. Remarkably, electrophoretic mobility shift assay and antibody supershift experiments indicated that all three major CRE-binding protein factors, i.e. CREB, ATF1, and CREM, may participate in forming specific DNA/protein complexes with the CRE of the TH gene. To address the transcriptional activation function of individual factors, we replaced the TH CRE with a GAL4-binding site and cotransfected this modified TH promoter-reporter gene with an effector plasmid that encodes GAL4-fused transcription factor. Our results indicate that CREB but not ATF1 can support basal promoter activity while both can robustly induce the promoter activity in response to co-expression of the catalytic subunit of cAMP-dependent protein kinase (PKA). We further show that the coactivator CBP up-regulates PKA-mediated activation of the TH promoter and, if tethered to the TH promoter by a GAL4-fusion, can robustly transactivate the TH promoter even in the absence of PKA. Collectively, our results suggest that multiple CRE-binding factors interact with the CRE and regulate, in conjunction with the coactivator CBP, the transcriptional activity of the TH gene.

Bacteriophage therapy for Staphylococcus aureus biofilm-infected wounds: a new approach to chronic wound care.

Background: Bacterial biofilms, which are critical mediators of chronic wounds, remain difficult to treat with traditional methods. Bacteriophage therapy against biofilm has not been rigorously studied in vivo. The authors evaluate the efficacy of a species-specific bacteriophage against Staphylococcus aureus biofilm–infected wounds using a validated, quantitative, rabbit ear model. Methods: Six-millimeter dermal punch wounds in New Zealand rabbit ears were inoculated with wild-type or mutant, biofilm-deficient S. aureus. In vivo biofilm was established and maintained using procedures from our previously published wound biofilm model. Wounds were left untreated, or treated every other day with topical S. aureus–specific bacteriophage, sharp débridement, or both. Histologic wound healing and viable bacterial count measurements, and scanning electron microscopy were performed following harvest. Results: Wild-type S. aureus biofilm wounds demonstrated no differences in healing or viable bacteria following bacteriophage application or sharp débridement alone. However, the combination of both treatments significantly improved all measured wound healing parameters (p < 0.05) and reduced bacteria counts (p = 0.03), which was confirmed by scanning electron microscopy. Bacteriophage treatment of biofilm-deficient S. aureus mutant wounds alone also resulted in similar trends for both endpoints (p < 0.05). Conclusions: Bacteriophages can be an effective topical therapy against S. aureus biofilm–infected wounds in the setting of a deficient (mutant) or disrupted (débridement) biofilm structure. Combination treatment aimed at disturbing the extracellular biofilm matrix, allowing for increased penetration of species-specific bacteriophages, represents a new and potentially effective approach to chronic wound care. These results establish principles for biofilm therapy that may be applied to several different clinical and surgical problems.

In vivo modeling of biofilm-infected wounds: a review.

Chronic wounds continue to represent a difficult and complex problem for both patients and healthcare providers. Bacterial biofilms represent a critical component of nonhealing wounds, utilizing several different mechanisms to inhibit innate inflammatory pathways and resist traditional therapeutics. Although in vitro biofilm systems have been well described and studied, understanding the intricacies of wound biofilm pathology requires appropriate in vivo models to understand the interactions between bacteria and host. In an effort to clarify the available literature, this review describes and critically evaluates all of the in vivo wound biofilm models currently published to-date, including model advantages and clinical applicability. We will also address the need for continued therapeutic development and testing using these currently available in vivo models.

Comparative Analysis of Single-Species and Polybacterial Wound Biofilms Using a Quantitative, In Vivo, Rabbit Ear Model

Introduction The recent literature suggests that chronic wound biofilms often consist of multiple bacterial species. However, without appropriate in vivo, polybacterial biofilm models, our understanding of these complex infections remains limited. We evaluate and compare the effect of single- and mixed-species biofilm infections on host wound healing dynamics using a quantitative, in vivo, rabbit ear model. Methods Six-mm dermal punch wounds in New Zealand rabbit ears were inoculated with Staphylococcus aureus strain UAMS-1, Pseudomonas aeruginosa strain PAO1, or both, totaling 10∧6 colony-forming units/wound. Bacterial proliferation and maintenance in vivo were done using procedures from our previously published model. Wounds were harvested for histological measurement of wound healing, viable bacterial counts using selective media, or inflammatory cytokine (IL-1β, TNF-α) expression via quantitative reverse-transcription PCR. Biofilm structure was studied using scanning electron microscopy (SEM). For comparison, biofilm deficient mutant UAMS-929 replaced strain UAMS-1 in some mixed-species infections. Results Bacterial counts verified the presence of both strains UAMS-1 and PAO1 in polybacterial wounds. Over time, strain PAO1 became predominant (p<0.001). SEM showed colocalization of both species within an extracellular matrix at multiple time-points. Compared to each monospecies infection, polybacterial biofilms impaired all wound healing parameters (p<0.01), and increased expression of IL-1β and TNF-α (p<0.05). In contrast, mixed-species infections using biofilm-deficient mutant UAMS-929 instead of wild-type strain UAMS-1 showed less wound impairment (p<0.01) with decreased host cytokine expression (p<0.01), despite a bacterial burden and distribution comparable to that of mixed-wild-type wounds. Conclusions This study reveals that mixed-species biofilms have a greater impact on wound healing dynamics than their monospecies counterparts. The increased virulence of polybacterial biofilm appears dependent on the combined pathogenicity of each species, verified using a mutant strain. These data suggest that individual bacterial species can interact synergistically within a single biofilm structure.

Deficient cytokine expression and neutrophil oxidative burst contribute to impaired cutaneous wound healing in diabetic, biofilm-containing chronic wounds.

Diabetic patients exhibit dysregulated inflammatory and immune responses that predispose them to chronic wound infections and the threat of limb loss. The molecular underpinnings responsible for this have not been well elucidated, particularly in the setting of wound biofilms. This study evaluates host responses in biofilm‐impaired wounds using the TallyHo mouse, a clinically relevant polygenic model of type 2 diabetes. No differences in cytokine or Toll‐like receptor (TLR) expression were noted in unwounded skin or noninoculated wounds of diabetic and wild‐type mice. However, diabetic biofilm‐containing wounds had significantly less TLR 2, TLR 4, interleukin‐1β, and tumor necrosis factor‐α expression than wild‐type wounds with biofilm (all p < 0.001). Both groups had similar bacterial burden and neutrophil infiltration after development of biofilms at 3 days postwounding, but diabetic wounds had significantly less neutrophil oxidative burst activity. This translated into a log‐fold greater bacterial burden and significant delay of wound epithelization for biofilm‐impaired diabetic wounds at 10 days postwounding. These results suggest that impaired recognition of bacterial infection via the TLR pathway leading to inadequate cytokine stimulation of antimicrobial host responses may represent a potential mechanism underlying diabetic susceptibility to wound infection and ulceration.

Topically Delivered Adipose Derived Stem Cells Show an Activated-Fibroblast Phenotype and Enhance Granulation Tissue Formation in Skin Wounds

Multipotent mesenchymal stem cells (MSCs) are found in various tissues and can proliferate extensively in vitro. MSCs have been used in preclinical animal studies and clinical trials in many fields. Adipose derived stem cells (ASCs) have several advantages compared to other MSCs for use in cell-based treatments because they are easy to isolate with relative abundance. However, quantitative approaches for wound repair using ASCs have been limited because of lack of animal models which allow for quantification. Here, we addressed the effect of topically delivered ASCs in wound repair by quantitative analysis using the rabbit ear model. We characterized rabbit ASCs, and analyzed their multipotency in comparison to bone marrow derived-MSCs (BM-MSCs) and dermal fibroblasts (DFs) in vitro. Topically delivered ASCs increased granulation tissue formation in wounds when compared to saline controls, whereas BM-MSCs or DFs did not. These studies suggest that ASCs and BM-MSCs are not identical, though they have similar surface markers. We found that topically delivered ASCs are engrafted and proliferate in the wounds. We showed that transplanted ASCs exhibited activated fibroblast phenotype, increased endothelial cell recruitment, and enhanced macrophage recruitment in vivo.

A direct role of the homeodomain proteins Phox2a/2b in noradrenaline neurotransmitter identity determination.

Development of noraderenergic (NA) neurons in the vertebrate brain is dependent on the homeodomain proteins Phox2a and 2b. Here, we show that Phox2a directly controls the NA identity by activating NA‐synthesizing dopamine β‐hydroxylase (DBH) gene. Single point mutations in the homeodomain of Phox2a resulted in a failure to transactivate the DBH promoter in vitro and resulted in the loss of NA neurons in vivo. In addition, injection of Phox2a‐specific antisense oligonucleotide induced the loss of NA neurons in developing zebrafish. Phox2a and 2b activate the DBH promoter and bind to three domains (PBD1–3). PBD1 is composed of two overlapping sites with which monomers of Phox2a can interact. In contrast, PBD2 and 3 interact with the dimeric form of Phox2a. Mutations in three or four, but not one or two, of the binding sites completely abolished activation of the DBH promoter by Phox2a or 2b, while the conversion of PBD3 to a consensus motif (ATTA) improved the DBH promoter activity by > 10‐fold. Taken together, these findings establish that Phox2a and 2b control the development of NA neurons in part by directly transactivating DBH transcription through interactions with four binding sites clustered in the proximal promoter.