Distinct cAMP Signaling Microdomains Differentially Regulate Melanosomal pH and Pigmentation.

cAMP signaling is a well-established regulator of melanin synthesis. Two distinct cAMP signaling pathways-the transmembrane adenylyl cyclase pathway, activated primarily by the MC1R, and the soluble adenylyl cyclase (sAC) pathway-affect melanin synthesis. The sAC pathway affects melanin synthesis by regulating melanosomal pH, and the MC1R pathway affects melanin synthesis by regulating gene expression and post-translational modifications. However, whether MC1R genotype affects melanosomal pH is poorly understood. We now report that loss of function MC1R does not affect melanosomal pH. Thus, sAC signaling appears to be the only cAMP signaling pathway that regulates melanosomal pH. We also addressed whether MC1R genotype affects sAC-dependent regulation of melanin synthesis. Although sAC loss of function in wild-type human melanocytes stimulates melanin synthesis, sAC loss of function has no effect on melanin synthesis in MC1R nonfunctional human and mouse melanocytes or skin and hair melanin in e/e mice. Interestingly, activation of transmembrane adenylyl cyclases, which increases epidermal eumelanin synthesis in e/e mice, leads to enhanced production of eumelanin in sAC-knockout mice relative to that in sAC wild-type mice. Thus, MC1R- and sAC-dependent cAMP signaling pathways define distinct mechanisms that regulate melanosomal pH and pigmentation.

Soluble adenylyl cyclase contributes to imiquimod-mediated inflammation and is a potential therapeutic target in psoriasis.

Cyclic AMP (cAMP) has a key role in psoriasis pathogenesis, as indicated by the therapeutic efficacy of phosphodiesterase inhibitors that prevent the degradation of cAMP. However, whether soluble adenylate cyclase (sAC) (encoded by the ADCY10 gene), which is an important source for cAMP, is involved in Th17 cell-mediated inflammation or could be an alternative therapeutic target in psoriasis is unknown. We have utilized the imiquimod model of murine psoriasiform dermatitis to address this question. Adcy10-/- mice had reduced erythema, scaling and swelling in the skin and reduced CD4+ IL17+ cell numbers in the draining lymph nodes, compared with wild-type mice after induction of psoriasiform dermatitis with imiquimod. Keratinocyte-specific knock out of Adcy10 had no effect on imiquimod-induced ear swelling suggesting keratinocyte sAC has no role in imiquimod-induced inflammation. During Th17 polarization in vitro, naive T cells from Adcy10-/- mice exhibited reduced IL17 secretion and IL-17+ T-cell proliferation suggesting that differentiation into Th17 cells is suppressed without sAC activity. Interestingly, loss of sAC did not impact the expression of Th17 lineage-defining transcription factors (such as Rorc and cMaf) but rather was required for CREB-dependent gene expression, which is known to support Th17 cell gene expression. Finally, topical application of small molecule sAC inhibitors (sACi) reduced imiquimod-induced psoriasiform dermatitis and Il17 gene expression in the skin. Collectively, these findings demonstrate that sAC is important for psoriasiform dermatitis in mouse skin. sACi may provide an alternative class of topical therapeutics for Th17-mediated skin diseases.

The potential impact of melanosomal pH and metabolism on melanoma.

Melanin is synthesized in melanocytes and is transferred into keratinocytes to block the effects of ultraviolet (UV) radiation and is important for preventing skin cancers including melanoma. However, it is known that after melanomagenesis and melanoma invasion or metastases, melanin synthesis still occurs. Since melanoma cells are no longer involved in the sun tanning process, it is unclear why melanocytes would maintain melanin synthesis after melanomagenesis has occurred. Aside from blocking UV-induced DNA mutation, melanin may provide other metabolic functions that could benefit melanoma. In addition, studies have suggested that there may be a selective advantage to melanin synthesis in melanoma; however, mechanisms regulating melanin synthesis outside the epidermis or hair follicle is unknown. We will discuss how melanosomal pH controls melanin synthesis in melanocytes and how melanosomal pH control of melanin synthesis might function in melanoma. We will also discuss potential reasons why melanin synthesis might be beneficial for melanoma cellular metabolism and provide a rationale for why melanin synthesis is not limited to benign melanocytes.

A nuclear cAMP microdomain suppresses tumor growth by Hippo pathway inactivation.

Cyclic AMP (cAMP) signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. Here, we present a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncover a nuclear cAMP microdomain that activates a tumor-suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397 and export of YAP from the nucleus with no change in YAP protein stability. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer; thus, drugs directed at the nuclear cAMP microdomain may provide avenues for the treatment of cancer.

Flow cytometric characterization of the hemocytes of blood cockles Anadara broughtonii (Schrenck, 1867), Anadara kagoshimensis (Lischke, 1869), and Tegillarca granosa (Linnaeus, 1758) as a biomarker for coastal environmental monitoring.

Marine bivalves are often used as a sentinel species in coastal environmental monitoring since changes in the environmental quality are often well preserved in their cells and tissues. Anadara and Tegillarca species of Arcidae, the blood cockles, are considered to be good sentinel species in monitoring coastal pollution and ecosystem health because they are distributed widely in the subsurface of intertidal mudflats. Internal cellular defense of the blood cockles to physical and biological stresses is mediated by the circulating hemocytes, while their hemocyte types and functions are poorly studied. In this study, we first characterized morphology and immune-related activities of hemocytes of three common blood cockles Anadara broughtonii, A. kagoshimensis, and Tegillarca granosa using flow cytometry. Based on cell morphology and immunological functions, we described five types of hemocytes identically in the three blood cockles: erythrocytes type-I (erythrocytes-I), erythrocytes type-II (erythrocytes-II), granulocytes, hyalinocytes, and blast-like cells. Erythrocytes were round cells containing hemoglobin with numerous granules in the cytoplasm and these cells consist of two central populations. Erythrocytes-I were the most abundant cells accounting for 80-89% of the total circulating hemocytes and exhibited a certain level of lysosome and oxidative capacity. Erythrocytes-II were the largest cells and displayed high lysosome content and the most active oxidative capacity. Both erythrocytes-I and erythrocytes-II did not show phagocytosis capacity. Granulocytes were intermediated-sized hemocytes characterized by granules in the cytoplasm and long pseudopodia on the cell surface, and these cells were mainly engaged in the cellular defense exhibiting the largest lysosome content, the most active phagocytosis, and high oxidative capacity. Contrary to granulocytes, hyalinocytes were comparatively small and round cells and exhibited no granules in the cytoplasm. Hyalinocytes displayed a certain level of lysosome and phagocytosis and oxidative capacities. Blast-like cells characterized by the smallest size and small quantity of cytoplasm and exhibited an absence of phagocytosis and extremely low oxidative capacity, suggesting that this population is not directly involved in the cell-mediated immune activities. In conclusion, flow cytometry indicated that three blood cockles had five types of hemocytes, and the erythrocytes and granulocytes were mainly involved in the immunological activities.

Biological Effects of the Herbal Plant-Derived Phytoestrogen Bavachin in Primary Rat Chondrocytes.

The aim of this study was to examine the anabolic and anticatabolic functions of bavachin in primary rat chondrocytes. With bavachin treatment, chondrocytes survived for 21 d without cell proliferation, and the proteoglycan content and extracellular matrix increased. Short-term monolayer culture of chondrocytes showed that gene induction of both aggrecan and collagen type II, major extracellular matrix components, was significantly upregulated by bavachin. The expression and activities of cartilage-degrading enzymes such as matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs were inhibited significantly by bavachin, while tissue inhibitors of metalloprotease were significantly upregulated. Bavachin inhibits the expression of inducible nitric oxide synthase, a representative catabolic factor, and downregulated the expression of nitric oxide, cyclooxygenase-2, and prostaglandin E2 in a dose-dependent manner in chondrocytes. Our results suggest that the bavachin has anabolic and potent anticatabolic biological effects on chondrocytes, which may have considerable promise in treating articular cartilage degeneration in the future.

Development of an Experimental Animal Model for Lower Back Pain by Percutaneous Injury-Induced Lumbar Facet Joint Osteoarthritis.

We report generation and characterization of pain-related behavior in a minimally invasive facet joint degeneration (FJD) animal model in rats. FJD was produced by a non-open percutaneous puncture-induced injury on the right lumbar FJs at three consecutive levels. Pressure hyperalgesia in the lower back was assessed by measuring the vocalization response to pressure from a force transducer. After hyperalgesia was established, pathological changes in lumbar FJs and alterations of intervertebral foramen size were assessed by histological and imaging analyses. To investigate treatment options for lumber FJ osteoarthritis-induced pain, animals with established hyperalgesia were administered with analgesic drugs, such as morphine, a selective COX-2 inhibitor, a non-steroidal anti-inflammatory drug (NSAID) (ketorolac), or pregabalin. Effects were assessed by behavioral pain responses. One week after percutaneous puncture-induced injury of the lumbar FJs, ipsilateral primary pressure hyperalgesia developed and was maintained for at least 12 weeks without foraminal stenosis. Animals showed decreased spontaneous activity, but no secondary hyperalgesia in the hind paws. Histopathological and microfocus X-ray computed tomography analyses demonstrated that the percutaneous puncture injury resulted in osteoarthritis-like structural changes in the FJs cartilage and subchondral bone. Pressure hyperalgesia was completely reversed by morphine. The administration of celecoxib produced moderate pain reduction with no statistical significance while the administration of ketorolac and pregabalin produced no analgesic effect on FJ osteoarthritis-induced back pain. Our animal model of non-open percutanous puncture-induced injury of the lumbar FJs in rats shows similar characteristics of low back pain produced by human facet arthropathy.

Radiologic evaluation of degeneration in isthmic and degenerative spondylolisthesis.

STUDY DESIGN: A cross-sectional imaging study. PURPOSE: The objective was to assess the degree of degeneration and the associated factors through imaging studies of the lesion segment and the adjacent superior and inferior segments of isthmic and degenerative spondylolisthesis. OVERVIEW OF LITERATURE: Few articles existed for degeneration and related factors in isthmic and degenerative spondylolisthesis. METHODS: The subjects were 95 patients diagnosed with spondylolisthesis. Simple plain radiographs including flexion and extension and magnetic resonance imaging were used to investigate the degree of translation, disc degeneration, high intensity zone (HIZ) lesion, Schmorl's node (SN) and Modic changes. RESULTS: Advanced disc degeneration, grade 5, was shown to be significant in the index segment of the isthmic type (p=0.034). Overall, type 2 Modic change was most common in both groups and also, it was observed more in the isthmus group, specifically, the index segment compared to the degenerative group (p=0.03). For the SN, compared to the degenerative type, the isthmus type had a significantly high occurrence in the index segment (p=0.04). For the HIZ lesions, the isthmus type had a higher occurrence than the degenerative type, especially in the upper segment (p=0.03). CONCLUSIONS: Most advanced disc degeneration, fifth degree, SN and Modic change occurred more frequently in the lesions of the isthmus type. HIZ lesions were observed more in the isthmus type, especially in the segment superior to the lesion.