Characterization of rhodanine derivatives as potential disease-modifying drugs for experimental mouse osteoarthritis.

OBJECTIVE: This study was performed to characterize selected rhodanine derivatives as potential preclinical disease-modifying drugs for experimental osteoarthritis (OA) in mice. METHODS: Three rhodanine derivatives, designated rhodanine (R)-501, R-502, and R-503, were selected as candidate OA disease-modifying drugs. Their effects were evaluated by intra-articular (IA) injection in OA mouse models induced by DMM (destabilization of the medial meniscus) or adenoviral overexpression in joint tissues of hypoxia-inducible factor (HIF)-2α or zinc importer ZIP8. The regulatory mechanisms impacted by the rhodanine derivatives were examined in primary-culture chondrocytes and fibroblast-like synoviocytes (FLS). RESULTS: All three rhodanine derivatives inhibited OA development caused by DMM or overexpression of HIF-2α or ZIP8. Compared to vehicle-treated group, for example, IA injection of R-501 in DMM-operated mice reduced median OARSI grade from 3.78 (IQR 3.00-5.00) to 1.89 (IQR 0.94-2.00, P = 0.0001). R-502 and R-503 also reduced from 3.67 (IQR 2.11-4.56) to 2.00 (IQR 1.00-2.00, P = 0.0030) and 2.00 (IQR 1.83-2.67, P = 0.0378), respectively. Mechanistically, the rhodanine derivatives inhibited the nuclear localization and transcriptional activity of HIF-2α in chondrocytes and FLS. They did not bind to Zn2+ or modulate Zn2+ homeostasis in chondrocytes or FLS; instead, they inhibited the nuclear localization and transcriptional activity of the Zn2+-dependent transcription factor, MTF1. HIF-2α, ZIP8, and interleukin-1ß could upregulate matrix-degrading enzymes in chondrocytes and FLS, and the rhodanine derivatives inhibited these effects. CONCLUSION: IA administration of rhodanine derivatives significantly reduced OA pathogenesis in various mouse models, demonstrating that these derivatives have disease-modifying therapeutic potential against OA pathogenesis.

Overexpression of secretory leukocyte peptidase inhibitor (SLPI) does not modulate experimental osteoarthritis but may be a biomarker for the disease.

OBJECTIVE: Osteoarthritic cartilage destruction can be regulated by the balance between proteases and anti-proteases. Here, we sought to identify novel cellular protease inhibitors associated with osteoarthritis (OA) pathogenesis. METHODS: Candidate molecules were screened from microarray data of chondrocytes treated with OA-associated catabolic factors. The functions of candidate molecules in OA pathogenesis were examined in primary-culture mouse articular chondrocytes and mouse models of OA, such as those stimulated by destabilization of the medial meniscus (DMM) or intra-articular (IA) injection of adenovirus expressing the candidate gene. The value of the selected candidate molecule as a biomarker of OA was examined by measuring its circulating levels in human and mouse blood. RESULTS: Bioinformatic analysis identified secretory leukocyte peptidase inhibitor (SLPI) as a highly upregulated cellular protease inhibitor in chondrocytes treated with pathogenic catabolic factors, including interleukin (IL)-1ß, hypoxia-inducible factor (HIF)-2α, and zinc importer ZIP8. The adenovirus-mediated overexpression of SLPI in joint tissues did not cause any OA-like change or modulate DMM- or HIF-2α-induced experimental OA in mice. SLPI also did not markedly modulate the expression of OA-associated catabolic or anabolic factors in chondrocytes. However, SLPI was specifically upregulated in OA cartilage, and the serum SLPI levels were significantly elevated in human OA patients and experimental OA mice, suggesting that SLPI may be a biomarker of OA. CONCLUSION: Although SLPI is upregulated in OA chondrocytes, it does not appear to per se modulate OA development in mice. However, it may be a potential biomarker of OA in humans and animal models.

Upregulation of lipocalin-2 (LCN2) in osteoarthritic cartilage is not necessary for cartilage destruction in mice.

OBJECTIVE: Lipocalin-2 (LCN2) is a recently characterized adipokine that is upregulated in chondrocytes treated with pro-inflammatory mediators and in the synovial fluid of osteoarthritis (OA) patients. Here, we explored the in vivo functions of LCN2 in OA cartilage destruction in mice. METHODS: The expression levels of LCN2 were determined at the mRNA and protein levels in primary cultured mouse chondrocytes and in human and mouse OA cartilage. Experimental OA was induced in wild-type (WT) or Lcn2-knockout (KO) mice by destabilization of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing hypoxia-inducible factor (HIF)-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or LCN2 (Ad-Lcn2). The effect of LCN2 overexpression on the cartilage of WT mice was examined by IA injection of Ad-Lcn2. RESULTS: LCN2 mRNA levels in chondrocytes were markedly increased by the pro-inflammatory cytokines, interleukin (IL)-1ß and tumor necrosis factor-α (TNF-α), and by previously identified catabolic regulators of OA, such as HIF-2α and components of the zinc-ZIP8-MTF1 axis. LCN2 protein levels were also markedly increased in human OA cartilage and cartilage from various experimental mouse models of OA. However, overexpression of LCN2 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Furthermore, LCN2 overexpression in mouse cartilage via IA injection of Ad-Lcn2 did not cause OA pathogenesis, and Lcn2 KO mice showed no alteration in DMM-induced OA cartilage destruction. CONCLUSIONS: Our observations collectively suggest that upregulation of LCN2 in OA cartilage is not sufficient or necessary for OA cartilage destruction in mice.

Upregulation of Atrogin-1/FBXO32 is not necessary for cartilage destruction in mouse models of osteoarthritis.

OBJECTIVE: In a preliminary study, we found that recently identified catabolic regulators of osteoarthritis (OA), including hypoxia-inducible factor (HIF)-2α and members of the zinc-ZIP8-MTF1 axis, upregulate the E3 ubiquitin ligase, Atrogin-1 (encoded by Fbxo32), in chondrocytes. As the ubiquitination/proteasomal degradation pathways are tightly regulated to modulate the expression of catabolic factors in chondrocytes, we examined the in vivo functions of Atrogin-1 in mouse models of OA. METHODS: The mRNA and protein levels of Atrogin-1 and other regulators of OA were determined in primary cultured mouse chondrocytes, OA human cartilage, and OA cartilage from wild-type (WT) and Fbxo32-knockout (KO) mice subjected to destabilization of the medial meniscus or intra-articular (IA) injection of adenoviruses expressing HIF-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or Atrogin-1 (Ad-Fbxo32). The effect of Atrogin-1 overexpression on the cartilage of WT mice was examined by IA injection of Ad-Fbxo32. RESULTS: Atrogin-1 mRNA levels in chondrocytes were markedly increased by treatment with interleukin-1ß, HIF-2α, and members of the zinc-ZIP8-MTF1 axis. Atrogin-1 protein levels were also increased in OA cartilage from humans and various mouse OA models. However, the forced overexpression of Atrogin-1 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Moreover, overexpression of Atrogin-1 in the mouse joint tissues failed to cause OA pathogenesis, and Fbxo32 knockout failed to affect post-traumatic OA cartilage destruction in mice. CONCLUSIONS: Although Atrogin-1 is upregulated in OA cartilage, overexpression of Atrogin-1 in the joint tissues or knockout of Fbxo32 does not affect OA cartilage destruction in mice.

Reciprocal activation of hypoxia-inducible factor (HIF)-2α and the zinc-ZIP8-MTF1 axis amplifies catabolic signaling in osteoarthritis.

OBJECTIVE: Hypoxia-inducible factor (HIF)-2α and the zinc-ZIP8-MTF1 axis in chondrocytes serve as catabolic regulators of osteoarthritic cartilage destruction by regulating the expression of catabolic factor genes. We explored possible crosstalk between these signaling pathways and its biological significance in osteoarthritis (OA). METHODS: Microarray analysis, various mRNA and protein assays were conducted using primary cultured mouse articular chondrocytes and experimental OA cartilage to reveal molecular mechanisms underlying the crosstalk between HIF-2α and the zinc-ZIP8-MTF1 axis. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or MTF1 (Ad-Mtf1) in wild-type mice or mice with cartilage-specific conditional knockout of HIF-2α (Epas1(fl/fl);Col2a1-Cre), ZIP8 (Zip8(fl/fl);Col2a1-Cre), or MTF1 (Mtf1(fl/fl);Col2a1-Cre). RESULTS: HIF-2α activated the zinc-ZIP8-MTF1 axis in chondrocytes by upregulating the Zn(2+) transporter ZIP8, thereby increasing Zn(2+) influx and activating the downstream transcription factor MTF1. The zinc-ZIP8-MTF1 axis, in turn, acted as a novel transcriptional regulator of HIF-2α. HIF-2α-induced activation of the zinc-ZIP8-MTF1 axis amplified HIF-2α regulation of OA cartilage destruction by synergistically promoting expression of matrix-degrading enzymes. Thus, HIF-2α-induced activation of the zinc-ZIP8-MTF1 axis, together with zinc-ZIP8-MTF1 regulation of HIF-2α, acted collectively to synergistically promote expression of matrix-degrading enzymes and OA cartilage destruction. CONCLUSION: Our findings identify a reciprocal activation mechanism involving HIF-2α and the zinc-ZIP8-MTF1 axis during OA pathogenesis that amplifies catabolic signaling and cartilage destruction.

Reciprocal regulation by hypoxia-inducible factor-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in osteoarthritis.

OBJECTIVE: Hypoxia-inducible factor-2α (HIF-2α) transcriptionally upregulates Nampt in articular chondrocytes. NAMPT, which exhibits nicotinamide phosphoribosyltransferase activity, in turn causes osteoarthritis (OA) in mice by stimulating the expression of matrix-degrading enzymes. Here, we sought to elucidate whether HIF-2α activates the NAMPT-NAD(+)-SIRT axis in chondrocytes and thereby contributes to the pathogenesis of OA. METHODS: Assays of NAD levels, SIRT activity, reporter gene activity, mRNA, and protein levels were conducted in primary cultured mouse articular chondrocytes. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1) or NAMPT (Ad-Nampt). The functions of SIRT in OA were examined by IA co-injection of SIRT inhibitors or adenovirus expressing individual SIRT isoforms or shRNA targeting specific SIRT isoforms. RESULTS: HIF-2α activated the NAMPT-NAD(+)-SIRT axis in chondrocytes by upregulating NAMPT, which stimulated NAD(+) synthesis and thereby activated SIRT family members. The activated NAMPT-SIRT pathway, in turn, promoted HIF-2α protein stability by negatively regulating its hydroxylation and 26S proteasome-mediated degradation, resulting in increased HIF-2α transcriptional activity. Among SIRT family members (SIRT1-7), SIRT2 and SIRT4 were positively associated with HIF-2α stability and transcriptional activity in chondrocytes. This reciprocal regulation was required for the expression of catabolic matrix metalloproteinases (MMP3, MMP12, and MMP13) and OA cartilage destruction caused by IA injection of Ad-Epas1 Ad-Nampt. CONCLUSION: The reciprocal regulation of HIF-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in OA cartilage destruction caused by HIF-2α or NAMPT.

Expression of RUNX3 in skin cancers.

BACKGROUND: Expression of Runt-related transcription factor 3 (RUNX3) is reduced in a large number of cancers. However, a few studies have reported higher expression of RUNX3 in several cancers, including basal cell carcinoma (BCC). In light of this, we explored the expression of RUNX3 in skin cancers generally, to determine whether it acts as an oncogene or a tumour-suppressor gene in skin tumours. AIM: To investigate the expression of RUNX3 in normal skin and malignant skin tumours. METHODS: RUNX3 expression was evaluated by western blotting in 24 specimens, comprising 6 malignant melanoma (MM), 6 squamous cell carcinoma (SCC), 6 BCC and 6 normal skin specimens. Immunohistochemical staining was carried out to analyse RUNX3 expression in 16 MM, 16 SCC and 16 BCC specimens. To identify where the protein was expressed, the cytoplasmic and nuclear protein expression of RUNX3 in skin cancer tissues was determined. A cell-proliferation study was performed on an MM line (G361) by small interfering (si)RNA transfection. RESULTS: The western blotting experiments showed that RUNX3 was not expressed in normal skin tissues, but it was overexpressed in all MM and SCC samples, and in five of the six BCC samples. Using immunochemistry, RUNX3 was found to be overexpressed in all cancer tissues analysed. Subcellular fraction analysis revealed that RUNX3 was expressed in the nuclei but not the cytoplasm of all the skin cancer tissues analysed, and RUNX3 silencing by siRNA in G361 cells resulted in a decrease in proliferation. CONCLUSIONS: Based on these results, we suggest that RUNX3 has an oncogenic potential and does not act as a tumour suppressor in skin cancers.

Dapsone hypersensitivity syndrome with circulating 190-kDa and 230-kDa autoantibodies.

Dapsone has potent anti-inflammatory effects, and is used in the treatment of leprosy, cutaneous vasculitis, neutrophilic dermatoses, and dermatitis herpetiformis and other blistering disorders. However, it may cause severe adverse reactions such as hypersensitivity syndrome, which is characterized by fever, skin rash, hepatitis and lymphadenopathy. We report a 44-year-old female Korean patient with dapsone hypersensitivity syndrome (DHS) that presented as a bullous skin eruption. The patient had a 1-year history of urticarial vasculitis, treated with antihistamines, prednisolone and dapsone. Although the skin lesions improved, she reported fever, nausea, abdominal pain, jaundice, fatigue and skin rashes. On physical examination, there were generalized erythematous macules and purpura with facial oedema that developed into vesicles on the upper limbs. Histological examination of a skin biopsy of a vesicular lesion found subepidermal oedema with a mixed inflammatory cell infiltrate, including eosinophils in the dermis. Indirect immunofluorescence testing using normal foreskin as substrate revealed IgG deposits in the basement membrane zone. Circulating autoantibodies against antigens of 190 and 230 kDa were found by immunoblotting analysis using epidermal extracts. This case illustrates DHS with the formation of circulating autoantibodies.

Relationship of protoporphyrin IX synthesis to photodynamic effects by 5-aminolaevulinic acid and its esters on various cell lines derived from the skin.

BACKGROUND: 5-Aminolaevulinic acid (ALA) and its esters act as precursors to the fluorescent photosensitizer protoporphyrin IX (PpIX) in photodynamic therapy (PDT). There is little information about how ALA and its esters induce PpIX synthesis and photodynamic effects in cell lines derived from the skin. OBJECTIVES: We compared the amount of PpIX synthesis induced by ALA and its esters in skin cell lines, and evaluated the relationship of PpIX synthesis to photodynamic effects by ALA and its esters in vitro. METHODS: Four cell lines, including human epidermal keratinocytes (HEK), human dermal fibroblast (hF), A431, and TXM13 were used. Cell survival was evaluated by the MTT assay. Fluorescence spectroscopy was used to measure the amount of PpIX synthesis induced by ALA and its esters. Flow cytometry measured cell death induced by ALA- and its esters-mediated PDT. RESULTS: ALA and its esters were not toxic at concentrations lower than 2 mmol L(-1) in all cell lines. PpIX synthesis was dose-dependent at low doses (0.01-0.1 mmol L(-1)), and ALA esters were more effective than ALA. Cell death occurred from necrosis rather than apoptosis just after light irradiation illumination on both ALA and its esters-treated cells. Cell death related more to PpIX synthesis than the irradiation light dose. CONCLUSIONS: PpIX production by ALA and its esters was induced on both normal and malignant cell lines derived from the skin, and cell death of PDT responses is closely related to the amount of PpIX synthesis rather than to the irradiation dose.

Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering.

HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.

Requirement for diacylglycerol and protein kinase C in HeLa cell-substratum adhesion and their feedback amplification of arachidonic acid production for optimum cell spreading.

Release of arachidonic acid (AA) and subsequent formation of a lipoxygenase (LOX) metabolite(s) is an obligatory signal to induce spreading of HeLa cells on a gelatin substratum (Chun and Jacobson, 1992). This study characterizes signaling pathways that follow the LOX metabolite(s) formation. Levels of diacylglycerol (DG) increase upon attachment and before cell spreading on a gelatin substratum. DG production and cell spreading are insignificant when phospholipase A2 (PLA2) or LOX is blocked. In contrast, when cells in suspension where PLA2 activity is not stimulated are treated with exogenous AA, DG production is turned on, and inhibition of LOX turns it off. This indicates that the formation of a LOX metabolite(s) from AA released during cell attachment induces the production of DG. Consistent with the DG production is the activation of protein kinase C (PKC) which, as with AA and DG, occurs upon attachment and before cell spreading. Inhibition of AA release and subsequent DG production blocks both PKC activation and cell spreading. Cell spreading is also blocked by the inhibition of PKC with calphostin C or sphingosine. The inhibition of cell spreading induced by blocking AA release is reversed by the direct activation of PKC with phorbol ester. However, the inhibition of cell spreading induced by PKC inhibition is not reversed by exogenously applied AA. In addition, inhibition of PKC does not block AA release and DG production. The data indicate that there is a sequence of events triggered by HeLa cell attachment to a gelatin substratum that leads to the initiation of cell spreading: AA release, a LOX metabolite(s) formation, DG production, and PKC activation. The data also provide evidence indicating that HeLa cell spreading is a cyclic feedback amplification process centered on the production of AA, which is the first messenger produced in the sequence of messengers initiating cell spreading. Both DG and PKC activity that are increased during HeLa cell attachment to a gelatin substratum appear to be involved. DG not only activates PKC, which is essential for cell spreading, but is also hydrolyzed to AA. PKC, which is initially activated as consequence of AA production, also increases more AA production by activating PLA2.

p38 kinase mediates nitric oxide-induced apoptosis of chondrocytes through the inhibition of protein kinase C zeta by blocking autophosphorylation.

This study investigated the molecular mechanisms underlying inhibition of protein kinase C (PKC) zeta by p38 kinase during nitric oxide (NO)-induced apoptosis of chondrocytes. Coimmunoprecipitation experiments showed that activation of p38 kinase following addition of an NO donor resulted in a physical association between PKCzeta and p38 kinase. Direct interaction of p38 kinase with PKCzeta was confirmed in vitro using p38 kinase and PKCzeta recombinant proteins. p38 kinase interacts with the regulatory domain of PKCzeta and its association blocked PKCzeta autophosphorylation. Micro LC-MS/MS analysis using recombinant proteins indicated that the interaction of p38 kinase with PKCzeta blocked autophosphorylation of PKCzeta on Thr-560, which is required for PKCzeta activation. Collectively, our results demonstrate a novel mechanism of PKCzeta regulation: following activation by the production of NO, p38 kinase binds directly to the PKCzeta regulatory domain, preventing PKCzeta autophosphorylation on Thr-560, thereby inhibiting PKCzeta activation.

Protein kinase A regulates chondrogenesis of mesenchymal cells at the post-precartilage condensation stage via protein kinase C-alpha signaling.

Chondrogenesis of mesenchymal cells during in vitro micromass culture requires the generation of cyclic adenosine monophosphate (cAMP) and subsequent activation of cAMP-dependent protein kinase A (PKA). In this study, we investigated the regulatory activity of PKA during chondrogenesis of chick limb bud mesenchymal cells. PKA activity was high in 1-day and 2-day cultures, which was followed by a slight decrease in 4-day and 5-day old cultures. Inhibition of PKA blocked chondrogenesis. It did not affect precartilage condensation, but it blocked the progression from the precartilage condensation stage to cartilage nodule formation. The PKA inhibition-induced blockage of chondrogenesis was accompanied by an altered expression of N-cadherin. Although expression of N-cadherin was detected during the early period of chondrogenesis, it became reduced as chondrogenesis proceeded. Still, inhibition of PKA maintained expression of N-cadherin throughout the micromass culture period. The inhibition of PKA did not affect expression of protein kinase C-alpha (PKCalpha), PKCepsilon, PKCdelta, and PKClambda/iota, which are the isoforms expressed in differentiating mesenchymal cells. However, PKA inhibition completely blocked activation of PKCalpha. Because PKC activity regulates N-cadherin expression and chondrogenesis, the PKA-mediated regulation of PKCalpha appears to be responsible for the PKA regulation of N-cadherin expression and chondrogenesis. Taken together, our results suggest that PKA regulates chondrogenesis by activating PKCalpha at the stage of post-precartilage condensation.

Integrin-mediated activation of mitogen-activated protein kinase is independent of the activation of protein kinase C epsilon during the spreading of HeLa cells on a gelatin substratum.

Spreading of HeLa cells on a gelatin substratum is initiated by the activation of protein kinase C epsilon (PKC epsilon) upon contact of the cells with the matrix. In this study, we examined the functional role of PKC epsilon in the activation of mitogen-activated protein kinase (MAP kinase) and its relationship to cell spreading. MAP kinase isoforms, Erk-1 and -2, are activated upon attachment of HeLa cells to gelatin. Inhibition of PKC with calphostin C blocked cell spreading without any effect on MAP kinase activation. In contrast, inhibition of MAP kinase kinase blocked adhesion-induced MAP kinase activation, but showed no effect on either translocation of PKC epsilon or cell spreading. Thus, activation of PKC epsilon that occurs upon HeLa cell attachment to gelatin is related to cell spreading but not to the activation of MAP kinase, and MAP kinase is activated upon HeLa cell attachment in the absence of cell spreading.

Regulation of chondrogenic differentiation of mesenchymes by protein kinase C alpha.

Chondrogenesis of chick limb bud mesenchymes requires the expression and activation of protein kinase C (PKC). This study was performed to identify PKC isoform(s) involved in the regulation chondrogenic differentiation of mesenchymes. Multiple PKC isoforms including alpha, epsilon, zeta and lambda/iota were expressed in mesenchymes derived from chick limb buds. Among the expressed PKC isoforms, the levels of PKC alpha and epsilon were increased during chondrogenic differentiation of mesenchymes. The increase in the expression of these isoforms is more evident in the particulate membrane fraction compared with the cytosolic fraction. Chondrogenesis was blocked by either selective inhibition or down-regulation of PKC alpha. In addition, the degree of chondrogenesis was closely correlated with the expression levels of PKC alpha but not other PKC isoforms expressed in mesenchymes. Thus, the results indicate that only PKC alpha is required for the induction of chondrogenic differentiation

Immunosuppressant rapamycin inhibits protein kinase C alpha and p38 mitogen-activated protein kinase leading to the inhibition of chondrogenesis.

Immunosuppressants are now known to modulate bone metabolism, including bone formation and resorption. Because cartilage, formed by differentiated chondrocytes, serves as a template for endochondral bone formation, we examined the effects of the immunosuppressant rapamycin on the chondrogenesis of mesenchymal cells and on the cell signaling that is required for chondrogenesis, such as protein kinase C, extracellular signal-regulated kinase-1 (ERK-1), and p38 mitogen-activated protein (MAP) kinase pathways. Rapamycin inhibited the expression of type II collagen and the accumulation of sulfate glycosaminoglycan, indicating inhibition of the chondrogenesis of mesenchymal cells. Rapamycin treatment did not affect precartilage condensation, but it prevented cartilage nodule formation. Exposure of chondrifying mesenchymal cells to rapamycin blocked activation of the protein kinase C alpha and p38 MAP kinase, but had no discernible effect on ERK-1 signaling. Selective inhibition of PKCalpha or p38 MAP kinase activity, which is dramatically increased during chondrogenesis, with specific inhibitors in the absence of rapamycin blocked the chondrogenic differentiation of mesenchymal cells. Taken together, our data indicate that the immunosuppressant rapamycin inhibits the chondrogenesis of mesenchymal cells at the post-precartilage condensation stage by modulating signaling pathways including those of PKCalpha and p38 MAP kinase.

Hypoxia-inducible factor-2α regulates Fas-mediated chondrocyte apoptosis during osteoarthritic cartilage destruction.

Apoptosis of articular chondrocytes is associated with the pathogenesis of osteoarthritis (OA). Recently, we demonstrated that hypoxia-inducible factor (HIF)-2α, encoded by Epas1, causes OA cartilage destruction by regulating the expression of various matrix-degrading enzymes. Here, we investigated the involvement of HIF-2α in chondrocyte apoptosis and OA cartilage destruction. HIF-2α levels in human and mouse OA chondrocytes were markedly elevated in association with increased apoptosis of articular chondrocytes. Overexpression or knockdown of HIF-2α alone did not cause chondrocyte apoptosis. However, HIF-2α expression markedly increased chondrocyte apoptosis in the presence of an agonistic anti-Fas (CD95) antibody. HIF-2α enhanced Fas expression and potentiated downstream signaling pathways, increasing the activity of initiator and executioner caspases. Overexpression of HIF-2α in mouse cartilage tissue, either by intra-articular injection of Epas1 adenovirus (Ad-Epas1) or in the context of chondrocyte-specific Epas1 transgenic mice, increased chondrocyte apoptosis and cartilage destruction. In contrast, chondrocyte-specific knockout of Epas1 in mice suppressed DMM (destabilization of the medial meniscus)-induced chondrocyte apoptosis and inhibited OA cartilage destruction. Moreover, Fas-deficient mice exhibited diminished chondrocyte apoptosis and OA cartilage destruction in response to Ad-Epas1 injection or DMM surgery. Taken together, our results demonstrate that HIF-2α potentiates Fas-mediated chondrocyte apoptosis, which is associated with OA cartilage destruction.

Serial changes in white matter lesions in a neonate with incontinentia pigmenti.

CASE REPORT: We report an infant who presented with clinical manifestations of incontinentia pigmenti (IP). Despite experiencing seizures in the early neonatal period, the patient had normal growth and development until recently. However, follow-up magnetic resonance imaging revealed sequential changes in white matter lesions. DISCUSSION: The pathogenesis of neurological involvement in IP has not been clearly elucidated and appears to be associated with various mechanisms, including developmental, destructive, and vascular processes. We have attempted to explain the pathogenesis of IP through these changes.

alpha-MSH inhibits TNF-alpha-induced matrix metalloproteinase-13 expression by modulating p38 kinase and nuclear factor kappaB signaling in human chondrosarcoma HTB-94 cells.

OBJECTIVE: Proinflammatory cytokine-induced expression of matrix metalloproteinases (MMPs) is a major cause of arthritic cartilage destruction. The neuropeptide, alpha-melanocyte-stimulating hormone (alpha-MSH), has been detected in the synovial fluid of arthritis patients, where it is thought to play an anti-inflammatory role. Here, we examined whether alpha-MSH acts via downregulation of MMP expression, and sought to elucidate the intracellular signal pathways underlying this effect. DESIGN: Human chondrosarcoma cell line, HTB-94 (SW1353) was pretreated with or without alpha-MSH and then treated with tumor necrosis factor-alpha (TNF-alpha). The effect of alpha-MSH on TNF-alpha-induced MMP-13 expression and mitogen-activated protein kinases' (MAPKs) activation were determined by reverse transcriptase-polymerase chain reaction and Western blot analysis. Additionally, the intracellular signaling of alpha-MSH was investigated using the inhibitors of MAPK and nuclear factor kappaB (NF-kappaB) and plasmids encoding dominant negative (dn) forms of inhibitor kappaB kinase-alpha (IKKalpha) and inhibitor kappaB kinase-beta (IKKbeta). RESULTS: We found that alpha-MSH pretreatment inhibited TNF-alpha-induced MMP-13 expression and p38 kinase phosphorylation in HTB-94 human chondrosarcoma cells. TNF-alpha-induced MMP-13 expression was not suppressed by extracellular signal-regulated kinase (ERK) inhibitors (PD98059 and U0126) or a c-jun terminal kinase (JNK) inhibitor (SP600125), but was inhibited by inhibitors of p38 kinase (SB203580) and NF-kappaB (SN-50 peptide) and dnIKKalpha and dnIKKbeta. CONCLUSIONS: Our results suggest that alpha-MSH regulates TNF-alpha-induced MMP-13 expression by decreasing p38 kinase phosphorylation and subsequent NF-kappaB activation in human chondrocytes and may be an effective inhibitor of MMP-13-mediated collagen degradation, providing new potential opportunities for the development of anti-arthritis therapeutics.

Differential translocation of protein kinase C epsilon during HeLa cell adhesion to a gelatin substratum.

The spreading of HeLa cells, following attachment to a collagen or gelatin substratum, requires the activation of protein kinase C (PKC). Membrane-bound PKC was previously shown to be activated during cell attachment and in response to the activation of a series of lipid second messengers turned on by the ligation of beta1-integrin collagen receptors. HeLa cells express the alpha, gamma, epsilon, zeta, lambda, and iota isozymes of PKC as determined by Western blotting with specific antibodies. Only PKCepsilon redistributed from the cytosol to the membrane during cell adhesion. Most of the PKCepsilon in cells that were in suspension was in the cytosolic fraction. During cell attachment to a gelatin matrix, all of the PKCepsilon moved out of the cytosol, with most going to the membrane fraction. After the cells became fully spread, PKCepsilon began to reappear in the cytosol. Translocation of PKCepsilon was not observed during the adhesion of cells to culture dishes where cells nonspecifically attach but do not spread. The conventional PKCalpha and -gamma isozymes were translocated from the cytosol to the membrane only when phorbol ester was present at a concentration that increases the rate and extent of cell spreading. Under normal conditions, i.e. in the absence of phorbol ester, PKCepsilon appears to be the PKC isozyme responsible for the regulation of HeLa cell adhesion to the extracellular matrix.