The Role Of DDR2 In Pulmonary Fibrosis And Its Role In TGF-? Signaling Pathway

Pulmonary fibros is the final stage of most interstitial lung diseases and the most common type of pulmonary fibrosis is the idiopathic pulmonary fibrosis with the cause still unknown.Most IPF patients died of respiratory failure at 3-5 years after diagnosis due to the lack of sensitive diagnosis in early stage and effective treatments.Therefore,the search for IPF diagnosis markers and the development of related target drugs have been a popular and difficult issue all over the world.The deposited of extracellular matrix(ECM),which is mostly consist of collagen,is the main pathological characteristics for pulmonary fibrosis diseases.Recent studies showed that ECM is not only the result of fibration but also one of the main reasons for the development of diseases.Four members of the integrin family and the discoidin domain receptors(including DDR1 and DDR2)make up the receptors on the surface of mammalian cell membranes that can mediate collagen signaling.Our former studies showed that DDR2 can promote lung fibrosis in a kinase activity-dependent manner and kinase activity-independent manner.And by suppressing the expression of DDR2 or its kinase activity can stop the progress of pulmonary fibrosis.Based on these studies,we put forward the following questions:(1)Whether is there any clinical significance in DDR2 associating with pulmonary fibrosis?(2)What is the molecular mechanism for DDR2 promoting pulmonary fibrosis in a kinase activity-independent manner?(3)Can the DDRs small molecular inhibitors use for the treatment of pulmonary fibrosis? In order to answer these questions,we have done the following studies.Study part one: The expression of discoidin domain receptor 2 in human interstitial lung diseases.Because other collagen receptors have been reported associated with pulmonary fibrosis as well,we investigated the expression of four members in the family of integrins(?1?1,?2?1,?10?1 and ?11?1)and two members in the family of discoidin domain receptor(DDR1 and DDR2)in clinical specimens of pulmonary fibrosis respectively.Quantitative real-time polymerase chain reaction(qPCR)was utilized to assess the mRNA expression.Immunoblot experiments were performed to analyze the protein abundance and activity.The tissue location was determined by immunohistochemical staining.qPCR data showed that it is only DDR2,other than the rest receptors,that whose mRNA displays the most dramatic difference between idiopathic pulmonary fibrosis(IPF)and healthy groups.The outstanding increases in DDR2 proteins were also observed in other types of interstitial lung diseases(ILD)besides IPF.DDR2-expressing cells in tissue sections of ILD were found to display spindle or fibroblastic shape.Study part two: The role of DDR2 in TGF-? signaling pathway.Transforming growth factor(TGF)-? is considered to be the most powerful driver of fibroblast ECM production and tissue fibrosis characterized to date.Drugs targeting TGF-? activation for IPF is in stage II clinical trials.However,TGF-? is also a cytokine with multi-effect.Even though the classic Smad pathway participate in many disease progressions,its function in regular cell activities is inevitable.Therefore,blocking the whole TGF-? pathway can raise the risk of canceration and Immunosuppression.Our former studies showed that deletion of DDR2 genes can affect the response of lung fibroblasts to TGF-? stimulation.In this part of study,we aimed at deeper explore the mechanism of interaction between DDR2 and TGF-?.We have confirmed that,the deficiency expression of DDR2 will not influence Smad pathway,but can suppress the activation of p38 and Akt.We also confirmed that the influence of DDR2 to these nonclassical pathways does not rely on the collagen induced tyrosine phosphorylation.So,is there a possibility for DDR2 to exert its influence on the TGF-? pathway by interacting with other proteins? We further investigated by immunoprecipitation / mass spectrometry and achieved initial progress.Study part three: The effect of two small molecule inhibitors,GZD856 and 6j on anti-fibrosisAlthough inflammatory response plays an important role in the initiation of fibrosis,clinical practice has demonstrated that anti-inflammatory therapy does not prevent the progression of pulmonary fibrosis.Most of time,when IPF patients have been diagnosed with high resolution CT the disease is usually in the advanced stage.The most common characteristic in this stage is the appearance of scattered fiber foci or reticular and honeycombing changes formed by fiber fusion.Since our first part of study has already confirmed that the dominant role of DDR2 in late stage of fibrosis,we confer that the positive feedback of ‘collagen stimulation-DDR2 activation-collagen synthesis' is the most important reason for the development of pulmonary fibrosis in advance stage and small molecule inhibitors targeting at DDR2 kinase activity can suppress this pathologic process.In this part of study,bleomycin-induced pulmonary fibrosis model of mice was used to examine the effect of two small molecule inhibitors.HE and Masson staining and measurement hydroxyproline content were carried out to evaluate the effect of inhibitor 6j.And the effect is rather remarkable.Also,we used Immunoblot to examine the effect of GZD856 and the result showed that under small dose of this inhibitor,the phosphorylated form of DDR2 and TAK1 were significantly decreased in vitro and combined with former animal experiments,GZD856 can effectively inhibit the expression of p-DDR2 and maintain significant inhibition against bleomyicin induced pulmonary fibrosis.