Melanoma shows highly aggressive behavior (i.e., local invasion and metastasis). Matrix metalloprotease-3 (MMP-3), a zinc-dependent endopeptidase, degrades several extracellular substrates and contributes to local invasion by creating a microenvironment suitable for tumor development. Here, we report that interleukin-1β (IL-1β) triggers the MMP-3 expression in canine melanoma cells. The activity of MMP-3 in the culture supernatant was increased in IL-1β-treated melanoma cells. IL-1β time- and dose-dependently provoked the mRNA expression of MMP-3. IL-1β induced the migration of melanoma cells; however, this migration was attenuated by UK356618, an MMP-3 inhibitor. When the cells were treated with the nuclear factor-κB (NF-κB) inhibitor TPCA-1, the inhibition of MMP-3 expression was observed. In IL-1β-treated cells, the phosphorylation both of p65/RelA and p105 was detected, indicating NF-κB pathway activation. In p65/RelA-depleted melanoma cells, IL-1β-mediated mRNA expression of MMP-3 was inhibited, whereas this reduction was not observed in p105-depleted cells. These findings suggest that MMP-3 expression in melanoma cells is regulated through IL-1β-mediated p65/RelA activation, which is involved in melanoma cell migration.

The rate of glucose uptake dramatically increases in cancer cells even in the presence of oxygen and fully functioning mitochondria. Cancer cells produce ATP by glycolysis rather than oxidative phosphorylation under aerobic conditions, a process termed as the “Warburg effect.” In the present study, we treated canine melanoma cells with the glucose analog 2-deoxy-D-glucose (2-DG) and investigated its effect on cell growth. 2-DG attenuated cell growth in a time- and dose-dependent manner. Cell growth was also inhibited following treatment with the glucose transporter (GLUT) inhibitor WZB-117. The treatment of 2-DG and WZB-117 attenuated the glucose consumption, lactate secretion and glucose uptake of the cells. The mRNA expression of the subtypes of GLUT was examined and GLUT1 and GLUT3 were found to be expressed in melanoma cells. The growth, glucose consumption and lactate secretion of melanoma cells transfected with siRNAs of specific for GLUT1 and GLUT3 was suppressed. These findings suggest that glucose uptake via GLUT1 and GLUT3 plays a crucial role for the growth of canine melanoma cells.

In autoimmune diseases, fibroblasts produce and secrete various cytokines and act as sentinel immune cells during inflammatory states. However, the contribution of sentinel immune cells (i.e. dermal fibroblasts) in autoimmune diseases of the skin, such as atopic dermatitis, has been obscure. The pro-inflammatory cytokine interleukin 1β (IL-1β) induces the expression of chemokines, such as interleukin 8 (IL-8), in autoimmune diseases of the skin. IL-8 induces the activation and recruitment of innate immune cells such as neutrophils to the site of inflammation. IL-1β-mediated induction of IL-8 expression is important for the pathogenesis of autoimmune diseases; however, the intracellular singling remains to be understood. To elucidate the mechanism of the onset of autoimmune diseases, we established a model for IL-1β-induced dermatitis and investigated MAPK signaling pathways in IL-1β-induced IL-8 expression. We also identified that a MAP3K Tpl2 acts as an upstream modulator of IL-1β-induced ERK1/2 activation in dermal fibroblasts. We observed an increase in the expression of IL-8 mRNA and protein in cells treated with IL-1β. ERK1/2 inhibitors significantly reduced IL-1β-induced IL-8 expression, whereas the inhibitor for p38 MAPK or JNK had no effect. IL-1β induced ERK1/2 phosphorylation, which was attenuated in the presence of an ERK1/2 inhibitor. IL-1β failed to induce IL-8 expression in cells transfected with siRNA for ERK1, or ERK2. Notably, a Tpl2 inhibitor reduced IL-1β-induced IL-8 expression and ERK1/2 phosphorylation. We confirmed that the silencing of Tpl2 in siRNA-transfected fibroblasts prevented both in IL-1β-induced IL-8 expression and ERK1/2 phosphorylation. Taken together, our data indicate the importance of Tpl2 in the modulation of ERK1/2 signaling involved in the IL-1β-induced development of autoimmune diseases affecting the dermal tissue, such as atopic dermatitis.

The specification of cell identity depends on the exposure of cells to sequences of bioactive ligands. All-trans retinoic acid (ATRA) affects neuronal development in the early stage, and it is involved in neuronal lineage reprogramming. We previously established a fibroblast-like dedifferentiated fat cells (DFATs) derived from highly homogeneous mature adipocytes, which are more suitable for the study of cellular reprogramming. Canine cognitive dysfunction is similar to human cognitive dysfunction, suggesting that dogs could be a pathological and pharmacological model for human neuronal diseases. However, the effect of ATRA on neuronal reprogramming in dogs has remained unclear. Therefore, in this study, we investigated the effect of ATRA on the neuronal reprogramming of canine DFATs. ATRA induced the expression of neuronal marker mRNA/protein. The neuron-like cells showed Ca2+ influx with depolarization (50 mM KCl; 84.75 ± 4.05%) and Na+ channel activation (50 μM veratridine; 96.02 ± 2.02%). Optical imaging of presynaptic terminal activity and detection of neurotransmitter release showed that the neuron-like cells exhibited the GABAergic neuronal property. Genome-wide RNA-sequencing analysis shows that the transcriptome profile of canine DFATs is effectively reprogrammed towards that of cortical interneuron lineage. Collectively, ATRA can produce functional GABAergic cortical interneuron-like cells from canine DFATs, exhibiting neuronal function with > 80% efficiency. We further demonstrated the contribution of JNK3 to ATRA-induced neuronal reprogramming in canine DFATs. In conclusion, the neuron-like cells from canine DFATs could be a powerful tool for translational research in cell transplantation therapy, in vitro disease modeling, and drug screening for neuronal diseases.

The pro-inflammatory cytokine interleukin 1β (IL-1β) induces the synthesis of prostaglandin E2 by upregulating cyclooxygenase-2 (COX-2) in the synovial tissue of individuals with autoimmune diseases, such as rheumatoid arthritis (RA). IL-1β-mediated stimulation of NF-κB and MAPK signaling is important for the pathogenesis of RA; however, crosstalk(s) between NF-κB and MAPK signaling remains to be understood. In this study, we established a model for IL-1β-induced synovitis and investigated the role of NF-κB and MAPK signaling in synovitis. We observed an increase in the mRNA and protein levels of COX-2 and prostaglandin E2 release in cells treated with IL-1β. NF-κB and ERK1/2 inhibitors significantly reduced IL-1β-induced COX-2 expression. IL-1β induced the phosphorylation of canonical NF-κB complex (p65 and p105) and degradation of IκBα. IL-1β also induced ERK1/2 phosphorylation but did not affect the phosphorylation levels of p38 MAPK and JNK. IL-1β failed to induce COX-2 expression in cells transfected with siRNA for p65, p105, ERK1, or ERK2. Notably, NF-κB inhibitors reduced IL-1β-induced ERK1/2 phosphorylation; however, the ERK1/2 inhibitor had no effect on the phosphorylation of the canonical NF-κB complex. Although transcription and translation inhibitors had no effect on IL-1β-induced ERK1/2 phosphorylation, the silencing of canonical NF-κB complex in siRNA-transfected fibroblasts prevented IL-1β-induced phosphorylation of ERK1/2. Taken together, our data indicate the importance of the non-transcriptional/translational activity of canonical NF-κB in the activation of ERK1/2 signaling involved in the IL-1β-induced development of autoimmune diseases affecting the synovial tissue, such as RA.

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of the immune response and inflammation. In this study, we investigated effect of the proinflammatory cytokine interleukin-1β (IL-1β) on IL-6 expression in canine dermal fibroblasts. IL-1β induced IL-6 mRNA expression and protein release in a time- and dose-dependent manner. When cells were treated with inhibitors of mitogen-activated protein kinases (MAPKs), the extracellular signal-regulated kinase (ERK) inhibitor FR180240 inhibited IL-1β-induced IL-6 mRNA expression, but not SP600125 or SKF86002, which are c-Jun N-terminal kinase (JNK) and p38 MAPK inhibitors, respectively. In cells treated with U0126, an inhibitor of MAPK/ERK kinase (MEK), which activates ERK, IL-1β-induced IL-6 mRNA expression was also inhibited. IL-1β stimulated ERK1/2 phosphorylation. In cells transfected with ERK1 and ERK2 isoform siRNAs, IL-1β-induced IL-6 mRNA expression was reduced. These observations suggest that IL-1β induces IL-6 expression via ERK1/2 signaling pathway in canine dermal fibroblasts.

Matrix metalloproteinases (MMPs) play a pivotal role in tissue remodeling by degrading the extracellular matrix (ECM) components. This mechanism is implicated in a variety of physiological and pathological cellular processes including wound healing. One of the key proteins involved in this process is the proinflammatory cytokine interleukin-1β (IL-1β, which induces the expression of MMP-3 mRNA and the secretion of MMP-3 protein by dermal fibroblasts. In this study, we first investigated the contribution of activating transcription factor 2 (ATF-2) to IL-1β-induced MMP-3 expression in dermal fibroblasts. Our results showed that in cells transfected with ATF-2 siRNA or treated with the ATF-2 inhibitor SBI-0087702, IL-1β-induced MMP-3 mRNA expression was reduced. We also demonstrated that IL-1β stimulates the phosphorylation of ATF-2. These observations suggest that ATF-2 plays an important role in IL-1β-induced MMP-3 expression. Next, we investigated the role of MAPK signaling in ATF-2 activation. In cells treated with the extracellular signal-regulated kinase (ERK) inhibitor FR180240, as well as in cells transfected with ERK1 and ERK2 siRNAs, IL-1β-induced MMP-3 mRNA expression was reduced. In addition, we showed that IL-1β induced the phosphorylation of ERK1/2. These observations suggest that ERK1 and ERK2 are involved in IL-1β-induced MMP-3 expression. However, ERK1 and ERK2 do seem to play different roles. While the ERK inhibitor FR180204 inhibited IL-1β-induced ATF-2 phosphorylation, only in cells transfected with ERK1 siRNA, but not ERK2 siRNA, IL-1β-induced ATF-2 phosphorylation was reduced. These findings suggest that the ERK1/ATF-2 signaling axis contributes to IL-1β-induced MMP-3 expression in dermal fibroblasts.

The proinflammatory mediator bradykinin stimulated cyclooxygenase-2 (COX-2) expression and subsequently prostaglandin E2 synthesis in dermal fibroblasts. The involvement of B2 receptors and Gαq in the role of bradykinin was suggested by using pharmacological inhibitors. The PKC activator PMA stimulated COX-2 mRNA expression. Bradykinin failed to induce COX-2 mRNA expression in the presence of PKC inhibitors, whereas the effect of bradykinin was observed in the absence of extracellular Ca2+. Bradykinin-induced COX-2 mRNA expression was inhibited in cells transfected with PKCε siRNA. These observations suggest that the novel PKCε is concerned with bradykinin-induced COX-2 expression. Bradykinin-induced PKCε phosphorylation and COX-2 mRNA expression were inhibited by an inhibitor of 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and bradykinin-induced PDK-1 phosphorylation was inhibited by phospholipase D (PLD) inhibitors, suggesting that PLD/PDK-1 pathway contributes to bradykinin-induced PKCε activation. Pharmacological and knockdown studies suggest that the extracellular signal-regulated kinase 1 (ERK1) MAPK signaling is involved in bradykinin-induced COX-2 expression. Bradykinin-induced ERK phosphorylation was attenuated in the cells pretreated with PKC inhibitors or transfected with PKCε siRNA. We observed the interaction between PKCε and ERK by co-immunoprecipitation experiments. These observations suggest that PKCε activation contributes to the regulation of ERK1 activation. Bradykinin stimulated the accumulation of phosphorylated ERK in the nuclear fraction, that was inhibited in the cells treated with PKC inhibitors or transfected with PKCε siRNA. Consequently, we concluded that bradykinin activates PKCε via the PLD/PDK-1 pathway, which subsequently induces activation and translocation of ERK1 into the nucleus, and contributes to COX-2 expression for prostaglandin E2 synthesis in dermal fibroblasts.

Inflammatory and microenvironmental factors produced by cancer cells are thought to directly or indirectly promote cancer cell growth. Prostaglandins, including prostaglandin E2, have key roles as a microenvironment factor in influencing the development of tumors, and are produced by the rate limiting enzyme cyclooxygenase 2 (COX-2). In this study, we used canine melanoma cells treated with the proinflammatory cytokine interleukin 1β (IL-1β) and investigated the transcriptional factor nuclear factor-κB (NF-κB) signaling in IL-1β-induced COX-2 expression. IL-1β induced prostaglandin E2 release and COX-2 mRNA expression in a time- and dose-dependent manner. In the cells treated with the NF-κB inhibitors BAY11-7082 and TPC-1, IL-1β-mediated prostaglandin E2 release and COX-2 mRNA expression were inhibited. IL-1β also provoked phosphorylation of p65/RelA and p105/NF-κB1, which are members of the NF-κB families. The IL-1β-induced phosphorylation of p65 and p105 was attenuated in the presence of both NF-κB inhibitors. In melanoma cells transfected with siRNA of p65 or p105, IL-1β-mediated COX-2 mRNA expression was inhibited. These findings suggest that canonical activation of NF-κB signaling plays a crucial role for inflammatory states in melanoma cells.

Tumor necrosis factor α (TNF-α) induces the expression and secretion of interleukin 8 (IL-8), which contributes to synovitis in rheumatoid arthritis (RA). To elucidate the mechanism of the onset of RA, we used synovial fibroblasts without autoimmune inflammatory diseases and investigated MAPK signaling pathways in TNF-α-induced IL-8 expression. Synovial fibroblasts isolated from healthy dogs were characterized by flow cytometry, which were positive for the fibroblast markers CD29, CD44, and CD90 but negative for the hematopoietic cell markers CD14, CD34, CD45, and HLA-DR. TNF-α stimulated the secretion and mRNA expression of IL-8 in a time- and dose-dependent manner. ERK and JNK inhibitors attenuated TNF-α-induced IL-8 expression and secretion. TNF-α induced the phosphorylation of ERK1/2 and JNK1/2. TNF-α-induced IL-8 expression was attenuated both in ERK2- and JNK1-knockdown cells. TNF-α-induced ERK1/2 or JNK1/2 was observed in ERK2- or JNK1-knockdown cells, respectively, showing that there is no crosstalk between ERK2 and JNK1 pathways. These observations indicate that the individual activation of ERK2 and JNK1 pathways contributes to TNF-α-induced IL-8 expression in synovial fibroblasts, which appears to be involved in the progress in RA.

ミニチュア・ダックスフント、12歳、去勢済み雄が間欠的な歩様異常を主訴に来院した。画像診断ならびに病理組織学検査より第12胸椎の孤立性骨形質細胞腫 (SOP) と診断された。ゾレドロン酸、メルファランならびにプレドニゾロンに極めてよく反応したが、2年後にゾレドロン酸の副作用と思われる下顎骨の骨壊死が発現した。本症例は、SOPが発生した犬に対するゾレドロン酸の副作用についての初報である。

A male neutered Abyssinian-crossbred cat presented with a chief complaint of head and truncal alopecia, and was diagnosed with Microsporum canis infection. Fluconazole was initially started, but was discontinued by the owner 70 days later. Dermatophytic granuloma developed in the dermis of the left lower jaw 22 months later. Although itraconazole was effective, the granuloma recurred subsequently, and spread to the oral cavity. The cat died from cachexia suspected to be caused by feeding difficulty on day 921 following the initial presentation. This report highlights the relevance of dermatophytosis and dermatophytic granuloma in cats, and the importance of compliance with medication on the part of pet owners.

Acute kidney injury (AKI) is characterized by a sudden loss of renal function. Early recognition of AKI, especially in critically ill patients, is essential for adequate therapy. Currently, neutrophil gelatinase-associated lipocalin (NGAL) is considered to be an effective biomarker of AKI; however, the regulation of its expression and function in renal tubular cells remains unclear. In this study, we investigated the regulation of the expression and function of NGAL in IL-1β-treated Madin-Darby canine kidney (MDCK) cells as a model of renal tubular cells. IL-1β induced a disturbance in the localization of E-cadherin and zonaoccludin-1 (ZO-1). The transepithelial electrical resistance (TER) also decreased 5 days after IL-1β treatment. IL-1β induced NGAL mRNA expression and protein secretion in a time- and dose-dependent manner, which occurred faster than the decrease in TER. In the presence of ERK1/2 and p38 inhibitors, IL-1β-induced NGAL mRNA expression and protein secretion were significantly attenuated. In the presence of recombinant NGAL, IL-1β-induced disturbance in the localization of E-cadherin and ZO-1 was attenuated, and the decrease in TER was partially maintained. These results suggest that NGAL can be used as a biomarker for AKI and that it functions as a protector from AKI.

Canine medial patellar luxation (MPL) is a very common orthopedic disease in small animals. Because the pathophysiology of this disease involves various pathways, the surgical techniques and results vary according to the veterinarian. Further, the landmark for block recession is not completely clear. We retrospectively evaluated 75 dogs (107 limbs) with MPL in whom our landmark for block recession was used from July 2008 to May 2013. Information regarding the breed, age, sex, body weight, body condition score (BCS), lateral vs bilateral, pre-operative grading, surgical techniques, removal of implants, concomitance with anterior cruciate ligament (ACL) rupture, re-luxation, re-operation, and rehabilitation was obtained from the medical records. The breeds were as follows: Chihuahua (n=23), Pomeranian (n=12), Yorkshire Terrier (n=9), and so on. The study group consisted of 33 males (castrated n=13) and 42 females (spayed n=21). The median age was 53.3±35.9 months (32-146 months); 13 cases were less than 12 months of age (17.3%). The pre-surgical BCSs were as follows: 1 (n=0), 2 (n=20), 3 (n=24), 4 (n=24) and 5 (n=7). The body weight was 4.51±3.48 kg (1.34-23.0 kg); 71 cases (94.7%) were less than 10 kg. The MPL grades (each limb) were G1 (n=1), G2 (n=18), G3 (n=78), and G4 (n=10); 32 cases were bilateral and 43 cases were unilateral (right n=27; left n=16). The specific surgical procedure (distal femoral osteotomy) was 3 stifles in Chihuahuas. Concurrent with ACL rupture was 16/107 stifles (15.0%) corrected with the over-the-top method or the extracapsular method in Papillons (5/6), Chihuahuas (5/23), and so on. The occurrences of re-luxation and re-operation were 3 out of 107 stifles (2.8%) and 0%, respectively. In this retrospective study, we present a potentially good surgical landmark for block recession of MPL in dogs.

Achilles tendon rupture is uncommon in small animal practice. A 9-month-old, female, mixed breed dog (weighing 2.2kg) was referred to our hospital with a primary complaint of right hind limb lameness. Complete right Achilles tendon rupture was diagnosed by physical examination and radiography. The tendon was surgically repaired the next day by using a three-loop and single near-far-far-near suture methods. Complete healing was achieved by 97 days post-surgery. This report describes the surgical technique used for complete Achilles tendon rupture repair in a young dog.

Feline hyperthyroidism (HT) is a common endocrine disorder worldwide, but clinical and laboratory features might vary geographically. The aim of this retrospective study was to evaluate feline HT in Japan, and compare results to those of previous study for feline HT. We evaluated 48 feline HT cases clinical and laboratory features. Surprisingly, the youngest patient was 32 months of age (2 year 9 months). There was no significant difference among the study subjects in sex, but frequency of spayed/castrated cats was high (85.4%). Median age was 186 months (32-272 months). 91.3% (n=42) of subjects were over 10 years of age, and 8.7% (n=4) were under 10 years of age. Clinical symptoms included vomiting, 56.3% (n=27); diarrhea, 2.1% (n=1); hyperactivity, 12.5% (n=6); emaciation, 41.7% (n=20); polyuria and polydipsia, 22.9% (n=11); chronic weight loss, 60.4% (n=29); and palpated enlarged thyroid, 2.1% (n=1). Concurrent findings included chronic kidney disease, 20.8% (n=10); congestive heart failure, 20.8% (n=10); tachycardia (over 240 beats/min), 18.8% (n=9); gallop rhythm, 31.3% (n=15); neurological disorders such as hind-limb paralysis, 14.6% (n=7); cystitis, 8.7% (n=4); gingivitis, 4.2% (n=2); diabetes mellitus, 4.2% (n=2); and arterial thromboembolism, 6.3% (n=3). In addition, laboratory features (complete blood counts and biochemistry) differed from those of previous reports in certain respects. Our results show that it might be important for practitioners to comprehend epidemiologic differences regarding feline HT worldwide.

Extracapsular technique with lateral fabellar suture (LFS) is one of the commonest surgical techniques for canine cranial cruciate ligament (CRCL) rupture. Among LFS methods, parallel double holes in the tibia are the most effective for the repair of canine stifle joint. The aim of this study was to evaluate the effectiveness of two-hole LFS for canine CRCL rupture. We retrospectively evaluated the outcome of canine CRCL rupture treated with two-hole LFS technique performed by two surgeons from October 2011 to September 2013. Breeds included Beagle (n=5), Yorkshire terrier (n=5), German shepherd (n=4), and so on. Mean body weight was 14.0±11.6 kg (>10 kg: n=16, <10 kg: n=22). Body condition score (5 scales) was as follows: 1 (n=0), 2 (n=1), 3 (n=18), 4 (n=18), and 5 (n=1). There were 18 males [neutered (n=11)] and 20 females [spayed (n=12)]. Mean age was 83.4±44.8 months (>12 months: n=35, <12 months: n=3). The type of rupture was partial (n=17) and complete (n=21). The affected site was right (n=20), left (n=18), and bilateral (n=4). Rehabilitation after operation was needed in 8 cases. There were 12 cases of previous medial patellar luxation operation, and 6 cases of opposite site CRCL surgery. Start of weight bearing and resolution of extension function was on day 1 (n=30), day 2 (n=0), and after day 2 (n=8). There was no evidence of surgical site infection/inflammation in any cases. The results of this study showed that two-hole LFS is an easy and effective technique for the repair of canine CRCL.

It has been known since many years that there are histamine receptors (H) in the heart. Histamines display chronotropic and inotropic activity, cardiovascular diseases, and are thought to be a systemic inflammatory disease. During heart failure, the histamine concentration is elevated. In addition, H2 blockers prolonged the survival period for human patients with heart failure. The aim of this study was to evaluate whether blood concentration of histamine is associated with canine valvular disease (CVD). The histamine concentrations of dogs with CVD are significantly higher than those of healthy dogs. The histamine concentration gradually increases during CVD and is highly correlated with the grade of heart murmur. In conclusion, the histamine concentration was higher in the population of dogs with CVD compared with the healthy controls. Although the etiopathogenesis of CVD is complex and incompletely understood, it likely involves histamine. Ultimately additional studies are required to determine whether histamine blockers might be useful for the management of dogs with cardiac valvular disease.

Diacetylated spermine, a polyamine, is clinically used as a tumor marker in human medicine. The purpose of this study was to evaluate the usefulness of diacetylated spermine as a nonspecific tumor marker in dogs. One hundred six dogs were randomly selected and divided into 4 groups (cancer-bearing, n=23; control, n=44; young, n=24; inflammatory, n=15). Urine samples were collected to measure diacetylated spermine and creatinine levels. The diacetylated spermine concentration was determined by enzyme-linked immunoassay, standardized by creatinine concentration. The urine diacetylated spermine concentration was markedly significant elevated in cancer-bearing dogs, as compared to that in control dogs (P=0.0081). Whilst the urine diacetylated spermine level in cancer-bearing group was not markedly different from that in the young group, there was significant different between levels in the control and young groups (P=0.019). No significant difference was observed between the inflammatory and cancer-bearing groups. It is suggested that urine diacetylated spermineis a useful tumor marker in dogs over 2 years of age.