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![Lemur Tyrosine Kinase 2 Regulates Fluid Transport in the Intestine için kapak resmi Lemur Tyrosine Kinase 2 Regulates Fluid Transport in the Intestine için kapak resmi](/client/assets/d79c3e4af2b6d196/ctx/images/no_image.png)
Lemur Tyrosine Kinase 2 Regulates Fluid Transport in the Intestine
Başlık:
Lemur Tyrosine Kinase 2 Regulates Fluid Transport in the Intestine
Yazar:
Dey, Isha, author.
ISBN:
9780438077928
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (174 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-11(E), Section: B.
Advisors: Neil A. Bradbury Committee members: Carl C. Correll; Charles W. Hutchins; Donghee Kim; Hector Rasgado-Flores.
Özet:
Lemur Tyrosine Kinase 2 (LMTK2) is an endogenous protein implicated in intracellular processes and in diseases; however, its physiological functions are poorly understood. It is a major component of the endosomal recycling pathway, whereby, proteins at the apical membrane are internalized by receptor-mediated endocytosis, and are recycled back to the apical membrane via endosomes, instead of degradation, owing to their long half-lives. One protein that undergoes the process of recycling is the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a Cl-- and HCO3 -- selective anion channel that functions at the apical membrane (as well as basolateral membrane in some tissues) of polarized epithelial cells. The channel is defective in cystic fibrosis, a life-threatening genetic disease that affects 1 in 3,400 births in the USA, and reduces life expectancy of to a maximum of ~38 years. At the molecular level, the disease is caused by loss of function mutations(s) in the cftr gene, which are manifested in the form of reduced CFTR transport and/or function at the apical membrane, leading to osmotic imbalance, mucus accumulation due to reduced clearance, and subsequent bacterial infection and inflammation. Thus, optimal functioning of CFTR is essential to maintain proper ion and fluid transport across the epithelium, thereby ensuring total body osmolality and homeostasis. One of the major organs that contribute to total body ionic homeostasis is the gut, where, the small intestine, expressing abundant CFTR, plays a major role in ion and fluid secretion. Studies in cell lines have shown that LMTK2 interacts with CFTR, and lack of LMTK2 impairs recycling of CFTR back to the plasma membrane after its internalization by clathrin-mediated endocytosis. This makes LMTK2 an important regulator of CFTR transport and hence CFTR-mediated ion and fluid transport, and also a therapeutic target for treating cystic fibrosis defects. However, to date, there are no known small molecule modulators of LMTK2 activity or expression. Moreover, studies in cell lines do not provide a very reliable recapitulation on the involvement of LMTK2 on CFTR function and hence fluid transport in-vivo. Thus, our objective in this study was to investigate the role of LMTK2 in intestinal fluid transport in a physiologically relevant model system. At the same time, considering the involvement of LMTK2 as a positive regulator of CFTR recycling, we wanted to characterize lmtk2 at the genetic level, to figure out if it is possible to regulate endogenous expression of the protein so as to increase overall activity of the protein.
Since the expression of a gene is regulated in part by different elements present in its promoter, we characterized lmtk2 gene upstream of its transcription start site, and identified a putative TPA-response element. As such, treating cells with low dose of TPA increased the promoter activity of lmtk2, leading to increase in transcript and protein levels. Moreover, TPA-response was mediated by transactivation and binding of the activator protein 1 (AP-1) transcription factor complex to the TPA-response element, leading to increase in LMTK2 expression. Thus, LMTK2 is a TPA-modulated gene.
At the physiological level, to study the role of LMTK2 in intestinal fluid transport, we developed self-renewing 3D intestinal organoids (also known as mini-guts) from mice, because they resemble the murine intestine with respect to cell polarity, cell type composition and organization, and thus, provide a faithful recapitulation of in-vivo functions under ex-vivo conditions. Using intestinal organoids from wild type and lmtk2 knockout mice of the same age, we demonstrated that not only does loss of lmtk2 gene product reduce total transepithelial fluid transport, it specifically reduces CFTR-mediated fluid transport into the intestinal lumen. Moreover, loss of LMTK2 alters expression of genes related to intestinal differentiation. On one hand, knocking out lmtk2 reduces expression of marker genes for intestinal stem cells, which are essential for growth and maintenance of the gut epithelium; while on the other hand, it increases marker gene expression for differentiated cell types.
Our study provides an insight into the characterization and physiological function of LMTK2 as a regulator of intestinal fluid transport and development. This knowledge will aid in the development of small molecule modulators of LMTK2 expression, as a therapeutic step towards increasing CFTR function and hence improving cystic fibrosis symptoms. Future studies with monitoring organ development over time, at the morphological as well as molecular levels, in presence or absence of LMTK2 would shed light on the precise involvement of LMTK2 in organ development and its potential as a therapeutic target for addressing developmental defects.
Notlar:
School code: 1489
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(694390.1) | 694390-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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