The Involvement Of RhoGDIα And Peripherin In The Cellular Redundancy Of Adrenal Medullary Chromaffin Cells Induced By NGF Via MAPK Signal Pathway

Background:Although bronchial asthma is a syndrome characterized by airway inflammation, airway hyperresponsiveness (AHR) and obstruction, an imbalance between nervous, endocrine and immune system also occurs in patients with asthma. Nerve growth factor (NGF) supports the development, survival and differentiation of sympathetic and sensory neurons. Recently, evidences have been accumulated that the increased NGF in patients with asthma attack triggers the functional redundancy of adrenal medullary chromaffin cells which are prone to differentiate towards nerve cells with their impaired endocrine functions, other than promotes airway inflammation and remodeling and aggravates the imbalance of immune response. In our previous studies, seventeen differential proteins were screened and identified based on proteomic technology. Our studies will focus on whether two of those proteins, peripherin and RhoGDIα, are involved in the cellular redundancy of chromaffin cells in vivo and in vitro. As known, NGF exerts its biological effects via activation of some intracellular signaling pathways such as mitogen-activated protein kinase (MAPK) pathway which plays a positive regulatory role in the NGF-induced differentiation of PC 12 (rat pheochromocytoma) cells. Therefore, we take chromaffin cells as target to investigate whether MAPK signal pathway is involved in the cellular redundancy of AMCC induced by NGF and mediates the expression of RhoGDIαand peripheirn in this process.Objective:To investigate whether RhoGDIαand peripherin are involved in the cellular redundancy primed by NGF in asthmatic rats via the regulation of NGF on the expression of those two proteins in primary AMCC, as well as those of anti-NGF on them in the adrenal medulla of asthmatic rats.Methods:The primary bovine AMCC were cultured and identified under contrast microscope and electronic speculum. The expression of RhoGDIαand peripherin was tested in the AMCC exposured to 100ng/ml NGF at different periods. The expression site of those two proteins in AMCC was shown through immunofluorescence.Forty eight Sprague-Dawley rats were randomly divided into four groups (control group, asthmatic group, NGF group and anti-NGF group, n=12). Sensitization and provocation (for asthmatic group, NGF group and anti-NGF group) were produced with OVA. The rats in NGF group and anti-NGF group were treated intraperitoneally with NGF or anti-NGF before every challenge. When all rats were killed, the lung and left adrenal tissues were fixed and embedded while the right adrenals were frozed quickly into liquid nitrogen. Pathological changes were observed in the lungs of rats of each groups. The expression of RhoGDIa and peripherin was detected in the adrenal tissues of rat models through immunohistochemical method and Western blot.Results:Observed with the invert phase-contrast microscope, the chromaffin cells were round with strong light refraction and prone to be scattered. The electron microscope showed interspersed chromaffin granules with obvious discrepancy in size and electron density in the cytoplasm. Immunofluorescent staining clearly showed that RhoGDIαwas predominantly localized to the cytosol as well as the plasma membrane of chromaffin cells cultured with NGF, while peripherin was only expressed in the cytosol of cells. The level of RhoGDIa and peripherin was expressed in an NGF-induced time-dependent manner. Western blot showed that the expression of RhoGDIa was strikingly lower and those of peripherin was notablely higher with NGF for more than 48h.The airway inflammation and remodeling response, including infiltration of inflammatory cells and thickening of alveolar septums and bronchial walls, was worsen in asthmatic and NGF group but markedly relieved in the anti-NGF group. Immunohistochemistry showed that there were many cells positive-immunoreactive to RhoGDIa or peripherin in the adrenal medullary tissues of asthmatic rats. The optical density (OD) of RhoGDIa in asthmatic group was higher than that in control (P<0.01), while that of peripherin was lower than that in control (P＜0.01). (The higher an OD shows, the lower a protein expression is). When compared with asthmatic group, the OD of RhoGDIαwas further elevated in NGF group (P<0.01) but depressed in anti-NGF group (P<0.01). In addition, there was markedly different OD of peripherin in NGF (P<0.01) and anti-NGF group (P<0.05) with comparision to asthmatic rats. Western blot showed that the relative expression of RhoGDIa in adrenal tissues was notablely lower in the asthmatic group than the control (P＜0.01). However, there is no significant difference of the expression level of the protein among the asthmatic, the NGF and the anti-NGF group (P>0.05). In addition, the peripherin expression of adrenal tissues is strikingly higher in the asthmatic group than the control (P＜0.01). However, it is significantly decreased in the anti-NGF group compared with the asthmatic group (P＜0.05), while there is no difference between the asthmatic and the NGF group (P>0.05).Conclusions:A reduced protein level of RhoGDIa and an augmented one of peripherin in the proteome map implies a decrease or increase expression in the adrenal medullary chromaffin cells of asthmatic rats. In a word, RhoGDIαand peripherin may be involved in the process of NGF-induced cellular redundancy in adrenal chromaffin cells of asthma models.Objective:To investigate whether the expression of RhoGDIa and peripheirn in the cellular redundancy of AMCC primed by NGF is mediated by MAPK signal pathway.Methods:The expression of p-ERK, p-JNK and p-p38 was tested by Western Blot in the AMCC exposed to 100ng/ml NGF for Omin,15min, 30min,60min and 90min respectively, and then the peak time of the phosphorylation of NGF-induced MAP kinases was determined. The AMCC were cultured with DMSO,50μM PD98059 (ERK inhibitor), 20μM SP600125 (JNK inhibitor) or 20μM SB202190 (p38 inhibitor) respectively for 30min followed by the inducement with 100ng/ml NGF. Both the cellular supernatant and the treated AMCC were collected at the peak time mentioned above, as well as 72h with NGF. The concentration of adrenaline (Ad) in the supernatant was tested with the corresponding ELISA kit and the ultrastructure of the treated AMCC was observed by electron microscope. The expression of RhoGDIαand peripherin in chromaffin cells in differently-treated groups was tested at 72h by Western blot.Results:The p-ERK, p-JNK and p-p38 in AMCC were expressed in the NGF-induced time-dependent manner. The expression of both p-ERK and p-JNK was up to the maximum at 15min, while that of p-p38 was peaked twice at 15min and 90min. On electron microscope it was showed that the chromaffin granules in the AMCC induced by NGF for 72h markedly decreased and gathered at the one side of the cells with the vacuolar degeneration in some mitochondrias. As a result, SB202190 had an ability to inhibit the effects of NGF on the ultrastructure of AMCC, but either PD98059 or SP600125 did not. Compared with the NGF group, the Ad concentration in the supernatant of AMCC cultured with NGF for 15min was not significantly changed among the three inhibitor-pretreated groups. When AMCC were exposured to NGF for 72h, the Ad of the PD+NGF group (4.65±0.36, P<0.01) was significantly decreased and that of the SB+NGF group (8.02±0.67, P＜0.01) was notably increased while that of the SP+NGF group (6.23±0.54, P>0.05) had no striking change, compared with the NGF group (6.88±0.77). Western blot showed, a decreased RhoGDIa level and an increased peripherin level in AMCC induced by NGF could be retarded by SB202190 instead of by PD98059 or SP600125.Conclusions:The p38/MAPK signal pathway may be involved in the cellular redundancy of AMCC induced by NGF and mediates the expression of RhoGDIαand peripherin in this process.