Investigation of endoplasmic reticulum stress in response to hypoxia in lung fibroblasts
(2020) MOBN02 20192Degree Projects in Molecular Biology
- Abstract
- Lack of oxygen delivery to the cells results in excessive production of oxygen free radicals in the cells leading to a pathological condition postulated as hypoxia. One of the most crucial transcription factors that can adapt to the fluctuating levels of hypoxia is Hypoxia Inducible Factor-1α (HIF-1α). Chronic obstructive pulmonary disease (COPD) is amongst the common lung diseases majorly caused by smoking that can exacerbate hypoxia. Smoking and oxidative stress tend to cause the accumulation of damaged proteins in the lungs leads towards endoplasmic reticulum (ER) stress, in response to that an adaptive mechanism known as “Unfolded protein response” is activated by the ER which activates stress mediators. This study was therefore... (More)
- Lack of oxygen delivery to the cells results in excessive production of oxygen free radicals in the cells leading to a pathological condition postulated as hypoxia. One of the most crucial transcription factors that can adapt to the fluctuating levels of hypoxia is Hypoxia Inducible Factor-1α (HIF-1α). Chronic obstructive pulmonary disease (COPD) is amongst the common lung diseases majorly caused by smoking that can exacerbate hypoxia. Smoking and oxidative stress tend to cause the accumulation of damaged proteins in the lungs leads towards endoplasmic reticulum (ER) stress, in response to that an adaptive mechanism known as “Unfolded protein response” is activated by the ER which activates stress mediators. This study was therefore designed to investigate the hypoxia stress and ER stress mediators by providing 1% oxygen (hypoxia) and 21% oxygen (normoxia) conditions to lung fibroblasts of healthy and COPD patient and effects were observed on mRNA and protein levels. Our study demonstrated that on RNA level, there was slightly increased tendency of HIF-1α (hypoxia inducible factor-1α) after culturing the lung fibroblasts for 4h in hypoxia as compared to normoxia.Whereas, in 24h the HIF-1α had increased expression during normoxia compared to hypoxia. However, an increased protein expression of HIF-1α was seen in healthy fibroblasts cultured for 24h in hypoxia than normoxia. Moreover, for COPD fibroblasts cultured for 4h, increased tendency of ER mediator ATF-6 (activating transcription factor-6), proteasomal subunit PSMA-1, antioxidant gene Nrf-2 (nuclear erythroid factor-2) and HIF-1α in both hypoxia and normoxia conditions as compared to the healthy fibroblasts was observed. Whereas fibroblasts cultured for 24h had increased levels of these genes in normoxia compared to hypoxia. Interestingly, distal lung fibroblasts had increased trend of HIF-1α and proteasomal proteins as compared to central fibroblasts. Furthermore, this study observed increased tendency of proliferation in hypoxia conditions than normoxia to adapt the stressed environment. Taken together, our analysis suggests that hypoxia induces detectable changes in the fibroblasts. Findings of this study might contribute to novel insights by inducing hypoxia to lung fibroblasts of smokers, non-smokers, and COPD patient to further observe the effects. (Less)
- Popular Abstract
- How do lung fibroblasts react in response to hypoxia?
Just as we could not survive in the room without oxygen, our body cells also feel stressed and unable to perform well in the absence of oxygen. Chronic obstructive lung disease (COPD) is a most common respiratory disease usually caused by tobacco smoking. Nevertheless, you could also contract the disease by inhaling the oxygen contaminated with environmental pollution even if you do not smoke.
Lung is the pinkish organ begins at the end of the trachea. An upside-down tree is formed by trachea and bronchi that is branched in small tubes called bronchioles followed by clusters of small air sacs called alveoli. The oxygen we breathe in passes from the trachea to the lungs, then... (More) - How do lung fibroblasts react in response to hypoxia?
Just as we could not survive in the room without oxygen, our body cells also feel stressed and unable to perform well in the absence of oxygen. Chronic obstructive lung disease (COPD) is a most common respiratory disease usually caused by tobacco smoking. Nevertheless, you could also contract the disease by inhaling the oxygen contaminated with environmental pollution even if you do not smoke.
Lung is the pinkish organ begins at the end of the trachea. An upside-down tree is formed by trachea and bronchi that is branched in small tubes called bronchioles followed by clusters of small air sacs called alveoli. The oxygen we breathe in passes from the trachea to the lungs, then transmitted to the organs of whole body through the blood. When there is not enough oxygen, all cells of the body get disturbed and there is a stress condition called hypoxia. Hypoxia worsens if it is followed by some airway disease like COPD. HIF-1α is an important factor which is activated in hypoxia. Endoplasmic reticulum (ER) is an organelle found inside the cell and is most sensitive to sense stress. ER acts as a machinery of producing, packaging, and shipping of proteins in the body. To adapt the awkward conditions going on in the body, ER serves as an adaptive mechanism system and releases proteins to cope up with the ongoing stress. Several genes get activated in response to the ongoing stress in the body.
In this project, PCR method was used to analyse the genes and immuno-fluorescent staining was done to visualize the proteins in the microscope. In our study, we provided 1% oxygen (hypoxia) and 21% oxygen (normoxia) conditions to lung fibroblasts to find out the genes which get activated in the lung fibroblasts. Interestingly, when we cultured healthy and COPD fibroblasts for 24h, a tendency of higher levels of hypoxia gene HIF-1α, anti-oxidative gene Nrf-2, ER stress mediators ATF-6, CHOP, protein homeostasis gene PSMB6, and extra-cellular matrix gene collagen-7 were seen in normoxia. Whereas proteins levels of HIF-1α were increased in hypoxia as compared to normoxia. However, after culturing of healthy fibroblasts for 4h, we observed a slight increased tendency of HIF-1α gene in hypoxia whereas COPD fibroblasts had increased expression of genes in both normoxia and hypoxia as compared to healthy fibroblasts in 4h.
To increase our knowledge in this vast array of disease, we need to continue working with genes by developing model systems which mimic the physiological atmosphere to get a better understanding of how lung diseases develop and grow. By doing that, we might be able to produce drug delivery systems that would be effective and targeted to the required area and could be important in cellular biology and chronic lung diseases. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9028752
- author
- Tahir, Rabeea Mirza
- supervisor
- organization
- course
- MOBN02 20192
- year
- 2020
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9028752
- date added to LUP
- 2020-09-09 14:45:27
- date last changed
- 2020-09-09 14:45:27
@misc{9028752,
abstract = {{Lack of oxygen delivery to the cells results in excessive production of oxygen free radicals in the cells leading to a pathological condition postulated as hypoxia. One of the most crucial transcription factors that can adapt to the fluctuating levels of hypoxia is Hypoxia Inducible Factor-1α (HIF-1α). Chronic obstructive pulmonary disease (COPD) is amongst the common lung diseases majorly caused by smoking that can exacerbate hypoxia. Smoking and oxidative stress tend to cause the accumulation of damaged proteins in the lungs leads towards endoplasmic reticulum (ER) stress, in response to that an adaptive mechanism known as “Unfolded protein response” is activated by the ER which activates stress mediators. This study was therefore designed to investigate the hypoxia stress and ER stress mediators by providing 1% oxygen (hypoxia) and 21% oxygen (normoxia) conditions to lung fibroblasts of healthy and COPD patient and effects were observed on mRNA and protein levels. Our study demonstrated that on RNA level, there was slightly increased tendency of HIF-1α (hypoxia inducible factor-1α) after culturing the lung fibroblasts for 4h in hypoxia as compared to normoxia.Whereas, in 24h the HIF-1α had increased expression during normoxia compared to hypoxia. However, an increased protein expression of HIF-1α was seen in healthy fibroblasts cultured for 24h in hypoxia than normoxia. Moreover, for COPD fibroblasts cultured for 4h, increased tendency of ER mediator ATF-6 (activating transcription factor-6), proteasomal subunit PSMA-1, antioxidant gene Nrf-2 (nuclear erythroid factor-2) and HIF-1α in both hypoxia and normoxia conditions as compared to the healthy fibroblasts was observed. Whereas fibroblasts cultured for 24h had increased levels of these genes in normoxia compared to hypoxia. Interestingly, distal lung fibroblasts had increased trend of HIF-1α and proteasomal proteins as compared to central fibroblasts. Furthermore, this study observed increased tendency of proliferation in hypoxia conditions than normoxia to adapt the stressed environment. Taken together, our analysis suggests that hypoxia induces detectable changes in the fibroblasts. Findings of this study might contribute to novel insights by inducing hypoxia to lung fibroblasts of smokers, non-smokers, and COPD patient to further observe the effects.}},
author = {{Tahir, Rabeea Mirza}},
language = {{eng}},
note = {{Student Paper}},
title = {{Investigation of endoplasmic reticulum stress in response to hypoxia in lung fibroblasts}},
year = {{2020}},
}