Dose dependent effect of LMW-HA on IL-1β induced MMP-1 and MMP-13

3. Results

3.3 Dose dependent effect of LMW-HA on IL-1β induced MMP-1 and MMP-13

Although HMW-HA could inhibit the induction effect of IL-1β on MMP-1 and MMP-13 expression, effects of LMW-HA on anti-inflammatory was not clear.

Therefore, SW1353 was pretreated with various concentrations (0, 0.1, 5, 10 μg/ml) of LMW-HA following with IL-1β treatment. However, not like HMW-HA, mRNA level of MMP-1 and MMP-13 were further increase after LMW-HA treatment (Figure 3). These finding could also be detected at protein level (Figure 5). The results indicated that LMW-HA may enhance the expression of inflammatory response mediator (MMPs) and induce cartilage matrix loss.

3.4 Dose dependent effect of HA on COX-2 expression

In the previous study, IL-1β was showed to activate clooxygenase-2 (COX-2) and increase prostaglandin E2 (PGE2) production., These would subsequently increase joint pain and further synovial inflammation[31]. Since HMW-HA has anti-inflammatory effect, the ability of HMW-HA on IL-1β activation was evaluated.

As shown in Figure 6, pretreated with HMW-HA would abolish the COX-2 expression that activated by IL-1β. This inhibitory effect could be detected in both RNA (Figure 6A) and protein level (Figure 6B).

In the pilot study, LMW-HA was showed to enhance the inflammatory mediator (i.e MMP-1 and MMP-13) production. It indicated that LMW-HA may enhance COX-2 expression at disease site. In order to figure out the effects of LMW-HA on COX-2, SW-1353 cells was pretreated with LMW-HA and then activated by IL-1β. As shown in Figure 7, the COX-2 expression level would be induced by IL-1β. Further induction could be detected via LMW-HA pretreatment.

3.5 Treatment of SW1353 with dose dependent manner of HMW-HA increase the level of HO-1 gene and protein expression

HO-1 is induced by a variety of stimuli or agents that cause oxidative stress, such as cytokines, reactive oxygen species, nitric oxide, heat shock, and hypoxia. HO-1 induction has also been shown to down-regulate the inflammatory response in animal models of acute inflammation [35]. But recently several reports have identified the heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) as a BCR/ABL-dependent survival molecule in CML cells and murine collagen-induced arthritis (CIA) model [37] [71]. Treatment with SnPP, which an inhibitor of HO-1, could significantly reduced the severity of CIA through inhibition of joint inflammation and cartilage destruction. We herein pretreated Chondrosarcoma SW1353 with HMW-HA to evaluate its effect on anti-inflammation. As data shown in Figure 8, SW-1353 express moderate amount of HO-1 and IL-1β treatment would not further inducing HO-1 expression. However, pretreated with HMW-HA would down regulate HO-1 expression at both RNA and protein level (Figure 8A and 8B).

In previous papers shown, HO-1 overexpression cannot slow the progression of the chronic inflammatory disease, whereas treatment with SnPP, which inhibits HO-1[38].

To examine the effects of LMW-HA on HO-1 expression, SW-1353 was pretreated with LMW-HA. Unlike the results obtained by HMW-HA treatment, insteated of down regulation, LMW-HA would enhance HO-1 expression in SW1353 cell (Figure 9).

3.6 Effects of HA on the expression of Nuclear receptor, PPARγ

Others have previously shown that PPARγ activators display anti-inflammatory and chondroprotective properties in vitro. However, the expression and regulation of PPARγ expression in cartilage are poorly defined. To determine the influence of

HMW-HA on the synthesis of MMP-13、MMP-1、COX-2、HO-1 and IL-1β in SW1353 of osteoarthritis (OA). As show in Figure 10(A) Figure 10 (B), Cell were preincubated with various concentrations of HMW-HA ( 0, 0.1, 0.3, 0.5 and 1 mg/mL ) for 30 minutes and then stimulated IL-1β 2 ng/mL for 24 hrs, After 24 hours, cell lysates were prepared and analyzed for peroxisome proliferator-activated receptor γ (PPARγ) protein expression by Immunoblotting. These data strongly suggest that HMW-HA with 1mg/mL increased PPARγ expression. According to Figure 10(A) Figure 10 (B) shown, to assess the contribution of these pathways in the HMW-HA-mediated upregulation of PPARγ, we found the MAPKs JNK and p38, but not ERK, are involved in HMW-HA-mediated upregulation of PPARγ. Taken together, Figure 12 shown, this data suggested that Mediation of HMW-HA-mediated upregulation of PPARγ by NF-κB inhibition.

As show in Figure 11(A), Figure 11(B). Cell were preincubated with various concentrations of LMW-HA ( 0, 0.1, 5, 10 μg/mL ) for 30 minutes and then stimulated IL-1β 2 ng/mL for 24 hrs. After 24 hours, cell lysates were prepared and analyzed for peroxisome proliferator-activated receptor γ (PPARγ) protein expression by Immunoblotting. These data suggest that LMW-HA decreased PPARγ expression through promoting phosphorylation of p38 MAPK pathway.

3.7 Effect of HMW-HA on phospho-Akt phosphorylation and IκBαexpression

According to pervious study, Glucosamine can induce p-Akt activation. To examine that HMW-HA can active pAkt the same as Glucosamine, so I used the maximum dose of HMW-HA, 1 mg/mL for time course. Figure 12(A) shown, we found HMW-HA can active IL-1β -induced pAkt activation.

To base on HMW-HA up-regulated Akt phosphorylation, we examine whether the

HMW-HA reduces inflammatory elements through up-regulated p-Akt and inhibited p- IκBα phosphorylation. we used the maximum dose of HMW-HA, 1 mg/mL for time course. Figure 12(B) shown, we found HMW-HA can inhibit IL-1β-induced p- IκBα phosphorylation after 30 minutes and inhibit total-IκBα degradation efficiently.

Figure 12(C)

3.8 Effect of HMW-HA on phosphorylation of MAPK pathways induced by IL-1β

In pervious papers shown, HA suppresses IL-1β-enhanced MMP-1 and MMP-3 synthesis in RSF (rheumatoid synovial fibroblasts) via ICAM-1 through down-regulation of NF-κB and p38 [21].

To examined the phosphorylation effect of HMW-HA, cells were pretreated HMW-HA 1 mg/mL for 30 minutes and then stimulated with IL-1β 2 ng/ml for different time course ( 0, 15, 30 and 60 min ) . Cell lysates is analyzed by immunoblotting. Figure 13(A) result that IL-1β is able to activate the phosphorylation of ERK starting at 15 min. Pretreatment with HMW-HA for 30 min, phosphorylation of ERK seems to slightly inhibited and lead to cell proliferation inhibited by HMW-HA at 15 min.

As in Figure 13(B) shown, this result the level of inflammatory protein secretion was decrease by treated HMW-HA and phosphorylation of JNK MAPK pathway, which pro-inflammtroy pathway was inhibited by HMW-HA at 60 minutes. This result was demonstrated that HMW-HA reduces inflammatory mediator through JNK MAPK pathway.

3.9 HMW-HA regulates cyclin D1/cdk4 protein expression of cell cycle

As in Figure 13(A), result that IL-1β is able to activate the phosphorylation of ERK starting at 15 min. Pretreatment with HMW-HA for 30 min, phosphorylation of ERK seems to slightly inhibited by HMW-HA as the same time as stimulated by IL-1β.

Hyaluronic acid were used in the clinical treatment of osteoarthritis, The result shown that HMW-HA had inhibitory effect of cyclin D1 and Cdk4 in dose-dependent manner and time-course. (Figure 14A&B)

3.10 Effect of LMW-HA affect phospho-Akt phosphorylation and IκBα expression

Recently papers shown, LMW-HA treatment decreased tumor cell proliferation, increased apoptosis, and downregulated activation of Akt and the expression of BCRP in glioma cells and treatment-resistant glioma stem cells [72]. To examine the effect of LMW-HA affect p-Akt in SW1353, I used the maximum dose of LMW-HA, 10 μg/mL for time course. Figure 15(A) shown, we found LMW-HA can down-regulate IL-1β-induced p-Akt activation and induced apoptosis in SW1353 the same as in glioma cells.

To determine whether LMW-HA induced p- IκBα phosphorylation and induced T- IκBα degradation. I used the maximum dose of LMW-HA, 10 μg/mL for time course.

Figure 15(B)(C) shown, we found LMW-HA can induce inflammatory elements through down-regulate p-Akt and induce NF-κB signaling pathway, after 15 minutes.

3.11 Effect of LMW-HA on phosphorylation of MAPK pathways induced by IL-1β

In pervious papers findings suggest that hyaluronan- CD44 interactions affect matrix metabolism via activation of NF-κB and p38 MAP kinase in C-28/I2 cell [73].

To determining that pro-inflammatory cytokine IL-1β as well as LMW-HA may be responsible for this up-regulation via a mechanism involving activation of the MAPKs (p38 and JNK) and NF-κB signaling pathways. In my poilt study konwn, LMW-HA can produce proinflammatory mediators, so we use different dose of LMW-HA ( 0, 0.1 5, 10 μg/mL ) treat chondrosarcoma SW1353 and detected by immunoblotting for different time course ( 0, 15, 30, 60 min ). As shown in Figure 16, LMW-HA is a strong inflammatory inducer for increasing p38 phosphorylation at 15 min.

4. Discussion

Osteoarthritis (OA) is a gradually progressing disorder of mammalian joints, characterised by the destruction of articular cartilage, which results in discomfort and dysfunction of the affected joint [1, 2]. The pathologic changes during the development of OA are remarkably similar and include proteoglycan degradation at the early stage, followed by type II collagen degradation, leading eventually to localized or complete loss of cartilage matrix [3].

Hyaluronan (HA) is a high molecular weight nonsulphated glycosaminoglycan (GAG) component of the extracellular matrix (ECM) present in many tissues. HA has many structural, rheological and physiological functions in tissues, including ECM and cellular interaction, growth factor intercaton, the regulation of osmosis and wound healing, immune response, re-epithelialixation stages [45]. Some HA plays several roles in the anti-inflammatory response, including the anti oxidant scavenging of ROS and other sources [21], these data suggest that, during oxidative stress and inflammation. Previously studies used high molecular weight HA and the opposite result was obtained. It is hypothesized that the inhibition of NF-κB DNA binding to the nucleus is probably the consequence of HA reduced ROS and MMPs production in the fibroblasts. NF-κB activation requires sequential phosphorylation, and degradation of IκBα, that, in the end, disappears from the cytoplasm.

According to these finding we use of HMW-HA (0.1-1 mg/mL) may reduce cell damage by inhibiting NF-κB as well as protecting cells from inflammatory mediators and MMP-1 and MMP-13 attack.

Hyaluronan oligosaccharides also activate NF-κB /I-κB α auto-regulatory loop [61], inducing transcription of metalloproteases MMP-9 and MMP-13 [43]. The induction of nitric-oxide synthase by such saccharides also occurs through a nuclear

NF-κB-dependent mechanism [62], and up-regulated JNK and p38 mitogen-activated protein kinase expression. According to these findings we also use HA oligosaccharides (0.1-10μg/mL) found it could induce MMP-1, MMP-13 and other inflammatory mediators, including COX-2 and HO-1 through induction of p38 MAP kinase and NF-κB pathway.

It has been proposed that production of VEGF after HO-1 overexpression in murine CIA ( Collagen-induced arthritis ) would promote angiogenesis. In the murine collagen-induced arthritis (CIA) model, HO-1 overexpression cannot slow the progression of the chronic inflammatory disease [38]. Our studies showed HO-1 expression during OA, whereas treatment with HMW-HA, which inhibits HO-1 expression [38]. It is interesting to note that recently observed a therapeutic effect of HO-1 inhibition via HMW-HA (0.1-0.5 mg/mL) in my study. The data presented here provide evidence to support the essential role of HO-1 in the negatively regulated anti-inflammatory function.

It was previously reported that the LMW-HA may induce injury by MMPs and others inflammatory mediators in both in vitro and in vivo [43]. According to these data, we suggest that LMW-HA (0.1-10 μg/mL) also could induce inflammatory mediators, including COX-2 and HO-1, the same as induce NF-κB pathway. The data also presented here provide evidence to support the essential role of HO-1 in the negatively regulated induce inflammatory function by injury.

PPARs are a family of nuclear hormone receptors that function as

ligand-activated transcription factors that upon heterodimerization with RXR, function as transcriptional regulators of glucose and lipid metabolism. PPARβ/δ is involved in development, wound healing, fatty-acid metabolism and repression of the

inflammatory response.

It has recently been discovered that PPARs are also strongly linked to inflammatory reaction. Inflammation inducers such as lipopolysaccharide (LPS) and tumor necrosis factor (TNFα) induce activation of NF-κB, a major transcription factor in the inflammatory process, and promote the secretion of a series of inflammatory cytokines in various cells. PPARα and PPARγ ligands can block the NF-κB pathway, modulating inflammatory reaction. We herein demonstrate that PPARγ exists expression by treatment HMW-HA (0.1-0.5 mg/mL), the same as increased anti-inflammatory after stimulation with IL-1β in SW1353. The data suggest that the ligand for PPARγ inhibit COX-2 and suppress inflammatory mediators. On the other hand, we also demonstrate that induction of inflammatory response by treatment with LMW-HA (0.1-10 μg/mL), and induction of NF-κB pathway.

It was previously reported that chondroitin-sulfate (CS) inhibited TNF-α-induced NF-κB activation and inducle nitric oxide synthase expression by blocking Akt/NF-κB signals in JB6 cells [74].

It has recently been discovered that the PI3K/Akt signaling pathway has been shown to play an important role in negatively regulating LPS-induce acute inflammatory responses in vitro and in vivo. Recently paper shown that α-Lipoic acid attenuates LPS-induced inflammatory responses by activating the PI3K /Akt signaling pathway in cultured human monocytic cells [75].

The PI3K/Akt pathway has been shown to act both positively and negatively on NF-κB-dependent pathway. These differences may be due to the use of different cell types and / or different stimulations. For example, in human monocytic cels, TLR-2-dependent PI3K/Akt pathway was shown to positively regulate the transactivation potential of p65. However, PI3K /Akt negatively regulated p65

transactivation induced by TLR4 activation. According to these data, we suggest that HMW-HA (1 mg/mL) time-course was able to activate the Akt phosphorylation and Inhibit NF-κB phosphorylation. We also demonstrate LMW-HA (10 μg/mL) time-course resulted from an enhance activation of NF-κB and p38 MAPK signaling cascade and a reduced activation of the PI3K/Akt pathway following LMW-HA treatment. Taken together, these data suggest that HA plays a critical role negatively regulating NF-κB activity by activation of the PI3K/Akt pathway.

In the current study, Peroxisome proliferator-activated receptor γ (PPARγ) ligands inhibit cell proliferation and induce apoptosis in cancer cells. Recently paper shown, troglitazone treatment, applied in a dose-dependent manner, cause a marked decrease in pRb, cyclin D1, cyclin D2, cyclin D3, Cdk2, Cdk4 and Cdk6 expression as well as significant increase in p21 and p27 expression in MDA-MB-231 cells [76]. In our study, we demonstrated that HMW-HA (0.1-1mg/mL) dose-dependent manner and time-course , resulted decrease in cyclin D1 and Cdk4 expression the same as promote cell cycle arrest in SW1353. Moreover, the PPARγ receptor has been shown to be implicated in carcinogenesis and inflammation. We herein suggested that the HMW-HA (0.1-0.5 mg/mL) inhibits in a dose-dependent manner IL-1β-mediated proinflammatory response by interfering with the phosphorylation of ERK1/2 inactivation.

CD44 is encoded by a single gene, but multiple isoforms of CD44 are generated by alternative RNA splicing. The gene for CD44 contains 20 exons. The nonvariant exons encode for an extracellular domain, a transmembrane domain, and an intracellular domain. Isoforms of CD44 are generated by the insertion of alternative exons (V1–V11) at a single site within the membrane-proximal portion of the extracellular domain The predominant 72–amino acid cytoplasmic domain can also be

replaced by an alternatively spliced shorter form. Differential posttranslational modifications, including glycosylation and the attachment of glycosaminoglycans, generate additional structural diversity of CD44.

The regulation of the affinity of cell adhesion molecules is prerequisite for regulating cell–cell and cell–matrix interactions mediated by broadly expressed receptors that are exposed continuously to their ligands. Most primary cells express CD44 but in a low affinity state that does not exhibit a capacity to bind to HA.

Cellular activation can induce a transition of CD44 to a high affinity state that mediates binding to HA. Transition from the “inactive” low affinity state to the

“active” high affinity state of CD44 on leukocytes can be induced by the ligation of antigen receptors, and on leukocytes and epithelial and other mesenchymal cells by soluble factors including cytokines [77, 78]. A variety of mechanisms have been implicated in the transition from inactive to active forms of CD44, including variant exon usage, receptor oligomerization, glycosylation, and sulfation [79]. However, to date, no data are available to indicate how these posttranslational modifications alter either the configuration of the receptor, its three-dimensional structure, or its molecular interactions with other moieties to modify the affinity of the receptor for HA.

In our study, we could not demonstrate that CD44 has high or low affinity state mediates binding to HMW-HA and / or LMW-HA, could not demonstrate that CD44 splicing form in SW1353, and could not demonstrate that CD44 receptor conformation change or posttranslation control (glycosylation, sulfation) by interacting with HMW-HA and /or LMW-HA.

In contrast to normal primary cells, many tumor-derived cells express CD44 in a high affinity state with capacity to mediate constitutive binding to HA. In addition to being a receptor for HA, CD44 can interact with several ECM proteins, such as

fibronectin and collagens, growth factors, cytokines and chemokines, as well as metalloproteinases [79], but less is known about the regulation of the interactions of these ligands with CD44. Transmembrane CD44 serves multiple roles, including mediating the metabolism of HA [80], in the regulation of tumor invasiveness and in the modulation of inflammatory cell function. Alterations in CD44 expression and structure have been documented in many types of cancer and are related to tumor dissemination [79]. Moreover, targeted deletion of CD44 prevented dissemination of some tumors [81]. Most of the known effects of CD44 on cell adhesion, migration, and metastasis are intimately associated with its capacity to promote cell attachment to HA. Recent findings suggest that CD44 might also promote metastasis through its association with other molecules. For example, CD44 provides a docking site for MMP-9 on the surface of melanoma and carcinoma cells [82] and thus can indirectly contribute to pericellular proteolysis to regulate tumor cell motility, growth factor activation, angiogenesis, as well as survival mechanisms.

In our study, may think that variable exons of CD44,or COOH terminal of CD44 or different amino acid binding site of CD44 variable exons, or HMW/LMW-HA interact with CD44 binding affinity might provide docking site with MMP-1, MMP-13 surface and LMW-HA of SW1353 and regulate proinflammatory response.

And in certain circumstances HMW-HA, that inhibited proinflammatory cytokine and LMW-HA inductuion of HA binding to CD44 in SW1353.

It has recently been discovered that the Ras signaling pathway is important in both cell proliferation and tumor progression. Alternatively spliced isoforms of CD44 containing variable exon 6 (v6) can serve as coreceptors for growth factor receptors that activate Ras. This paper shown that use v6-specific small interfering RNA (siRNA) to investigate the role of CD44 alternative splicing in Ras signaling. Authors identify a positive feedback loop in which Ras signaling promotes CD44v6 splicing,

and CD44v6 then sustains late Ras signaling, which is important for cell cycle progression. These results are the first demonstration of a positive feedback loop linking signaling-dependent alternative splicing to mitogenic progression. The production of CD44 variants through alternative splicing is regulated by splicing factors, such as Sam68 and SRm160, and stimulated by Ras/MAPK (mitogenactivated protein kinase) signaling (Ras–Raf–MEK–ERK). These splicing factors depend upon exon splicing enhancers in the CD44 variable exons.

In our study, we were not demonstrate that CD44 variants by some splincing factors in SW1353, and we do not know that splicing factors stimulated by Ras/MAPK signaling.

Their activity in promoting the inclusion of CD44 variable exons is controlled by Ras/MAPK signaling, at least in part through modification of splicing factors at the level of phosphorylation. However, the signaling pathway between Ras/MAPK

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