COX-2 expression and cell cycle progression in human fibroblasts

Cyclooxygenase-2 (COX-2) is continuously expressed in mostcancerous cells where it appears to modulate cellular proliferation andapoptosis. However, little is known about the contribution oftransient COX-2 induction to cell cycle progression or programmed celldeath in primary cells. In this study we determined whether COX-2regulates proliferation or apoptosis in human fibroblasts. COX-2 mRNA, protein, and prostaglandin E₂(PGE₂) were not detected in quiescent cells but wereexpressed during the G₀/G₁ phase of the cellcycle induced by serum. Inhibition of COX-2 did not alter G₀/G₁ to S phase transition or induceapoptosis at concentrations that diminished PGE₂.Addition of interleukin-1 to serum enhanced COX-2 expression andPGE₂ synthesis over that by serum alone but had no effecton the progression of these cells into S phase. Furthermore,platelet-derived growth factor drove the G₀ fibroblasts into the cell cycle without inducing detectable levels of COX-2 orPGE₂. Collectively, these data show that transient COX-2expression in primary human fibroblasts does not influence cell cycle progression.

     

Myricetin blocks lipoteichoic acid-induced COX-2 expression in human gingival fibroblasts

Periodontitis is an infectious disease caused by microorganisms present in dental bacterial plaque. Lipoteichoic acid (LTA) is a component of the external membrane of Gram-positive bacteria. It causes septic shock. Ingested flavonoids have been reported to directly affect the regulation of cyclooxygenase-2 (COX-2) expression induced by bacterial toxins. In this study, we examined the effects of four flavonoids (luteolin, fisetin, morin and myricetin) on the activation of ERK1/2, p38 and AKT, and on the synthesis of COX-2 in human gingival fibroblasts treated with LTA from Streptococcus sanguinis. We found that luteolin and myricetin blocked AKT and p38 activation and that myricetin blocked LTA-induced COX-2 expression. The results of our study are important for elucidating the mechanism of action of flavonoid regulation of inflammatory responses.     

iNOS signaling interacts with COX-2 pathway in colonic fibroblasts

COX-2 and iNOS are two major inflammatory mediators implicated in colorectal inflammation and cancer. Previously, the role of colorectal fibroblasts involved in regulation of COX-2 and iNOS expression was largely ignored. In addition, the combined interaction of COX-2 and iNOS signalings and their significance in the progression of colorectal inflammation and cancer within the fibroblasts have received little investigation. To address those issues, we investigated the role of colonic fibroblasts in the regulation of COX-2 and iNOS gene expression, and explored possible mechanisms of interaction between COX-2 and iNOS signalings using a colonic CCD-18Co fibroblast line and LPS, a potential stimulator of COX-2 and iNOS. Our results clearly demonstrated that LPS activated COX-2 gene expression and enhanced PGE(2) production, stimulated iNOS gene expression and promoted NO production in the fibroblasts. Interestingly, activation of COX-2 signaling by LPS was not involved in activation of iNOS signaling, while activation of iNOS signaling by LPS contributed in part to activation of COX-2 signaling. Further analysis indicated that PKC plays a major role in the activation and interaction of COX-2 and iNOS signalings induced by LPS in the fibroblasts.     

Helicobacter pylori gamma-glutamyltranspeptidase upregulates COX-2 and EGF-related peptide expression in human gastric cells

Gastric mucosa responds to Helicobacter pylori-induced cell damage by increasing the expression of COX-2 and EGF-related peptides. We sought to investigate the bacterial virulence factor/s and the host cellular pathways involved in the upregulation of COX-2, HB-EGF and amphiregulin in MKN 28 and AGS gastric mucosal cells. H. pylori strain CCUG 17874 was grown in Brucella broth supplemented with 0.2% (2,6-dimethyl)-beta-cyclodextrins. The soluble proteins released in the culture medium by the bacterium were fractionated by exclusion size and anion exchange chromatography. A single peak retaining the ability to upregulate COX-2 and HB-EGF mRNA and protein expression was obtained. SDS-PAGE analysis of the peak showed two peptides with an apparent molecular weight of 38 and 22 kDa, which were identified by automated Edman degradation analysis as the N-terminal and C-terminal peptides of H. pylori gamma-glutamyltranspeptidase respectively. Acivicin, a selective gamma-glutamyltranspeptidase inhibitor, counteracted H. pylori-induced upregulation of COX-2 and EGF-related peptide mRNA expression. An H. pylori isogenic mutant gamma-glutamyltranspeptidase-deficient strain did not exert any effect on COX-2, HB-EGF and amphiregulin mRNA expression. Blockade of phosphatidylinositol-3 kinase and p38 kinase, but not MAP kinase kinase, inhibited H. pylori gamma-glutamyltranspeptidase-induced upregulation of COX-2 and EGF-related peptide mRNA expression.     

Ca2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts

Fibroblasts isolated from jaw cysts expressed calcium-sensing receptor (CasR). In the fibroblasts elevated extracellular Ca(2+) ([Ca(2+)](o)) increased fluo-3 fluorescence intensity, and the production of inositol(1,4,5)trisphosphate and active protein kinase C. Phospholipase C inhibitor U-73122 attenuated the Ca(2+)-induced increase in fluo-3 fluorescence intensity. Elevated [Ca(2+)](o) enhanced the expression of cyclooxygenase-2 (COX-2) mRNA and protein, and the secretion of prostaglandin E(2) in the fibroblasts. CasR activator neomycin also increased the expression of COX-2 mRNA, and U-73122 attenuated the Ca(2+)-induced expression of COX-2 mRNA. Elevated [Ca(2+)](o)-induced phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and U-73122 inhibited the Ca(2+)-induced phosphorylation. The inhibitors for each kinase, PD98059, SB203580, and SP600125, attenuated the Ca(2+)-induced expression of COX-2 mRNA. These results suggest that in jaw cyst fibroblasts elevated extracellular Ca(2+) may enhance COX-2 expression via the activation of ERK1/2, p38 MAPK, and JNK through CasR.     

Aromatase and COX-2 expression in human breast cancers

We have investigated aromatase and the inducible cyclooxygenase COX-2 expression using immunocytochemistry in tumors of a series of patients with advanced breast cancer treated with aromatase inhibitors. Aromatase was expressed in 58/102 breast cancers. This is similar to the percentage previously reported for aromatase activity. Interestingly, aromatase was expressed in a variety of cell types, including tumor, stromal, adipose, and endothelial cells. Since prostaglandin E₂ is known to regulate aromatase gene expression and is the product of COX-2, an enzyme frequently overexpressed in tumors, immunocytochemistry was performed on the tissue sections using a polyclonal antibody to COX-2. Aromatase was strongly correlated (P<0.001) with COX-2 expression. These results suggest that PGE₂ produced by COX-2 in the tumor may be important in stimulating estrogen synthesis in the tumor and surrounding tissue. No correlation was observed between aromatase or COX-2 expression and the response of the patients to aromatase inhibitor treatment. However, only 13 patients responded. Nine of these patients were aromatase positive. Although similar to responses in other studies, this low response rate to second line treatment suggests that tumors of most patients were no longer sensitive to the effects of estrogen. Recent clinical studies suggest that greater responses occur when aromatase inhibitors are used as first line treatment. In the intratumoral aromatase mouse model, expression of aromatase in tumors is highly correlated with increased tumor growth. First line treatment with letrozole was effective in all animals treated and was more effective than tamoxifen in suppressing tumor growth. Letrozole was also effective in tumors failing to respond to tamoxifen, consistent with clinical findings. In addition, the duration of response was significantly longer with the aromatase inhibitor than with tamoxifen, suggesting that aromatase inhibitors may offer better control of tumor growth than this antiestrogen.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

PAF is involved in the Mycoplasma arthritidis superantigen-triggering pathway for iNOS and COX-2 expression in murine peritoneal cells

We investigated the capacity of Mycoplasma arthritidis mitogen (MAM) to induce (a) expression of the inducible enzymes cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS), (b) production of prostaglandin E2 (PGE2) and nitric oxide (NO), and (c) involvement of platelet-activating factor (PAF) in the MAM-induced activation pathway. Resident peritoneal cells from C3H/HePas mice were incubated with MAM in the presence or absence of a PAF-antagonist (WEB2170) or COX-2 inhibitors (nimesulide or NS398). Enzyme expression was evaluated by immunoblotting, PGE2 by EIA, and NO by Griess reaction. Following MAM-stimulation of peritoneal cells, expression of COX-2 was detected at 3 h (peak levels at 12 h) and of iNOS at 6 h (peak levels at 20 h). PGE2 increased till 20 h, decreasing thereafter, whereas NO increased with time. WEB2170 (5 x 10(-5) M) treatment caused 44% inhibition of NO output and reduced iNOS expression (48% at the peak of expression). Concomitant treatment with WEB2170 and nimesulide (10(-5) M) reversed these inhibitory effects. WEB2170 reduced COX-2 expression (43% at the peak of expression) and prevented the decline in PGE2 levels after 20 h. These results suggest the involvement of PAF in the signaling pathway triggered by MAM that leads to expression of iNOS and COX-2, and show that PAF regulates the production of NO, possibly by controlling levels of PGE2.     

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E2 (PGE2) or leukotriene B4 (LTB4) by subchondral osteoblasts, PGE2 levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE2 to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB4 levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plus transforming growth factor-beta (TGF-beta), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)2D3 and TGF-beta also modulated PGE2 production. TGF-beta stimulated PGE2 production in both OA osteoblast groups, whereas 1,25(OH)2D3 alone had a limited effect but decreased the effect of TGF-beta in the low OA osteoblasts subgroup. This modulation of PGE2 production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE2 levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE2 to LTB4 is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)2D3 and TGF-beta depending on their endogenous low and high PGE2 levels.     

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E₂ (PGE₂) or leukotriene B₄ (LTB₄) by subchondral osteoblasts, PGE₂ levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE₂ to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB₄ levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) plus transforming growth factor-β (TGF-β), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)₂D₃ and TGF-β also modulated PGE₂ production. TGF-β stimulated PGE₂ production in both OA osteoblast groups, whereas 1,25(OH)₂D₃ alone had a limited effect but decreased the effect of TGF-β in the low OA osteoblasts subgroup. This modulation of PGE₂ production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE₂ levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE₂ to LTB₄ is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)₂D₃ and TGF-β depending on their endogenous low and high PGE₂ levels.     

Regulation and function of COX-2 gene expression in isolated gastric parietal cells

We examined expression, function, and regulation of the cyclooxygenase (COX)-2 gene in gastric parietal cells. COX-2-specific mRNA was isolated from purified (>95%) canine gastric parietal cells in primary culture and measured by Northern blots using a human COX-2 cDNA probe. Carbachol was the most potent inducer of COX-2 gene expression. Gastrin and histamine exhibited minor stimulatory effects. Carbachol-stimulated expression was inhibited by intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (90%), protein kinase C (PKC) inhibitor GF-109203X (48%), and p38 kinase inhibitor SB-203580 (48%). Nuclear factor (NF)-kappaB inhibitor 1-pyrrolidinecarbodithioic acid inhibited carbachol-stimulated expression by 80%. Similar results were observed in the presence of adenoviral vector Ad.dom.neg.IkappaB, which expresses a repressor of NF-kappaB. Addition of SB-203580 with Ad.dom.neg.IkappaB almost completely blocked carbachol stimulation of COX-2 gene expression. We examined the effect of carbachol on PGE(2) release by enzyme-linked immunoassay. Carbachol induced PGE(2) release. Ad.dom.neg.IkappaB, alone or with SB-203580, produced, respectively, partial (70%) and almost complete (>80%) inhibition of carbachol-stimulated PGE(2) production. Selective COX-2 inhibitor NS-398 blocked carbachol-stimulated PGE(2) release without affecting basal PGE(2) production. In contrast, indomethacin inhibited both basal and carbachol-stimulated PGE(2) release. Carbachol induces COX-2 gene expression in the parietal cells through signaling pathways that involve intracellular Ca(2+), PKC, p38 kinase, and activation of NF-kappaB. The functional significance of these effects seems to be stimulation of PGE(2) release.     

Cytokine-mediated PGE2 expression in human colonic fibroblasts

We investigatedprostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84,HT-29, and LS 174T) and the effect of PGE₂ on fibroblast morphology.Cytokine-stimulated PGE₂production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) proteinand mRNA expression were evaluated. BasalPGE₂ levels were low in all celltypes (0.15-6.47 ng/mg protein). Treatment for 24 h with interleukin-1 (IL-1; 10 ng/ml) or tumor necrosis factor- (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions ofPGE₂ synthesis in CCD-18Cocultures and similar results in primary fibroblast cultures; maximalinductions with IL-1 in colonic epithelial cell lines were from zeroto fivefold. Treatment of CCD-18Co fibroblasts with IL-1 causedmaximal 21- and 53-fold increases, respectively, in PGHS-2 protein andmRNA levels without altering PGHS-1 expression.PGE₂ (0.1 µmol/l) elicited adramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and targetfor colonic prostanoids in vivo.

     

Inhibition of both COX-1 and COX-2 is required for development of gastric damage in response to nonsteroidal antiinflammatory drugs.

We examined the gastric ulcerogenic property of selective COX-1 and/or COX-2 inhibitors in rats, and investigated whether COX-1 inhibition is by itself sufficient for induction of gastric damage. Animals fasted for 18 h were given various COX inhibitors p.o., either alone or in combination, and they were killed 8 h later. The nonselective COX inhibitors such as indomethacin, naproxen and dicrofenac inhibited PG production, increased gastric motility, and provoked severe gastric lesions. In contrast, the selective COX-2 inhibitor rofecoxib did not induce any damage in the stomach, with no effect on the mucosal PGE(2) contents and gastric motility. Likewise, the selective COX-1 inhibitor SC-560 also did not cause gastric damage, despite causing a significant decrease in PGE(2) contents. The combined administration of SC-560 and rofecoxib, however, provoked gross damage in the gastric mucosa, in a dose-dependent manner. SC-560 also caused a marked gastric hypermotility, whereas rofecoxib had no effect on basal gastric motor activity. On the other hand, the COX-2 mRNA was expressed in the stomach after administration of SC-560, while the normal gastric mucosa expressed only COX-1 mRNA but not COX-2 mRNA. These results suggest that the gastric ulcerogenic property of conventional NSAIDs is not accounted for solely by COX-1 inhibition and requires the inhibition of both COX-1 and COX-2. The inhibition of COX-1 up-regulates the COX-2 expression, and this may counteract the deleterious influences, such as gastric hypermotility and the subsequent events, due to a PG deficiency caused by COX-1 inhibition.     

Vitamin D receptor expression is linked to cell cycle control in normal human keratinocytes

To improve our understanding of the cutaneous vitamin D system, we studied vitamin D receptor (VDR) gene regulation in cultured human keratinocytes. Because VDR and its ligand 1 alpha,25-dihydroxyvitamin D(3) have been implicated in epidermal growth control, we investigated VDR expression as related to cellular proliferation by using different cell cycle synchronization protocols. Keratinocytes, deprived of growth factors, were forced into quiescence and a concomitant loss of VDR expression was observed. Mitogenic stimulation of these G(0) cells however quickly upregulated VDR levels several hours ahead the G(1)-S transition point. Growth arrest at the G(1)-S border by mimosine treatment or at the metaphase by nocodazole also downregulated VDR levels but a restoration of VDR expression was again quickly achieved after reentering the cell cycle. These findings indicate that VDR expression in keratinocytes is restricted to actively cycling cells, but not limited to one particular phase of the cell cycle.     

cAMP-dependent proteolysis of GATA-6 is linked to JNK-signaling pathway

A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK.     

AMP-activated protein kinase is involved in COX-2 expression in response to ultrasound in cultured osteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. Cyclooxygenase-2 (COX-2) is a crucial mediator in mechanically induced bone formation. AMP-activated protein kinase (AMPK) has reported to sense and regulate the cellular energy status in various cell types. Here we found that US-mediated COX-2 expression was attenuated by LKB1 and AMPKalpha1 small interference RNA (siRNA) in human osteoblasts. Pretreatment of osteoblasts with AMPK inhibitor (araA and compound C), p38 inhibitor (SB203580), NF-kappaB inhibitor (PDTC), IkappaB protease inhibitor (TPCK) and NF-kappaB inhibitor peptide also inhibited the potentiating action of US. US increased the kinase activity and phosphorylation of LKB1, AMPK and p38. Stimulation of osteoblasts with US activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. US-mediated an increase of IKKalpha/beta activity, kappaB-luciferase activity and p65 and p50 binding to the NF-kappaB element was inhibited by araA, SB203580 and LKB1 siRNA. Our results suggest that US increased COX-2 expression in osteoblasts via the LKB1/AMPKalpha1/p38/IKKalphabeta and NF-kappaB signaling pathway.     

L-type Ca2+ channel function is linked to dystrophin expression in mammalian muscle

Background

In dystrophic mdx skeletal muscle, aberrant Ca²⁺ homeostasis and fibre degeneration are found. The absence of dystrophin in models of Duchenne muscular dystrophy (DMD) has been connected to altered ion channel properties e.g. impaired L-type Ca²⁺ currents. In regenerating mdx muscle, ‘revertant’ fibres restore dystrophin expression. Their functionality involving DHPR-Ca²⁺-channels is elusive.

Methods and Results

We developed a novel ‘in-situ’ confocal immuno-fluorescence and imaging technique that allows, for the first time, quantitative subcellular dystrophin-DHPR colocalization in individual, non-fixed, muscle fibres. Tubular DHPR signals alternated with second harmonic generation signals originating from myosin. Dystrophin-DHPR colocalization was substantial in wt fibres, but diminished in most mdx fibres. Mini-dystrophin (MinD) expressing fibres successfully restored colocalization. Interestingly, in some aged mdx fibres, colocalization was similar to wt fibres. Most mdx fibres showed very weak membrane dystrophin staining and were classified ‘mdx-like’. Some mdx fibres, however, had strong ‘wt-like’ dystrophin signals and were identified as ‘revertants’. Split mdx fibres were mostly ‘mdx-like’ and are not generally ‘revertants’. Correlations between membrane dystrophin and DHPR colocalization suggest a restored putative link in ‘revertants’. Using the two-micro-electrode-voltage clamp technique, Ca²⁺-current amplitudes (i_max) showed very similar behaviours: reduced amplitudes in most aged mdx fibres (as seen exclusively in young mdx mice) and a few mdx fibres, most likely ‘revertants’, with amplitudes similar to wt or MinD fibres. Ca²⁺ current activation curves were similar in ‘wt-like’ and ‘mdx-like’ aged mdx fibres and are not the cause for the differences in current amplitudes. i_max amplitudes were fully restored in MinD fibres.

Conclusions

We present evidence for a direct/indirect DHPR-dystrophin interaction present in wt, MinD and ‘revertant’ mdx fibres but absent in remaining mdx fibres. Our imaging technique reliably detects single isolated ‘revertant’ fibres that could be used for subsequent physiological experiments to study mechanisms and therapy concepts in DMD.