Abstract
The ubiquitous hyaladherin, hyaluronan-binding protein 1 (HABP1/p32/gC1qR) upon stable overexpression in normal fibroblasts (F-HABP07) has been reported to induce mitochondrial dysfunction, growth retardation and apoptosis after 72 h of growth. HABP1 has been observed to accumulate in the mitochondria resulting in generation of excess Reactive Oxygen Species (ROS), mitochondrial Ca++ efflux and drop in mitochondrial membrane potential. In the present study, autophagic vacuolation was detected with monodansylcadaverin (MDC) staining from 36 h to 60 h of culture period along with elevated level of ROS in F-HABP07 cells. Increased expression of autophagic markers like MAP-LC3-II, Beclin 1 and autophagic modulator, DRAM confirmed the occurrence of the phenomenon. Reduced vacuole formation was observed upon treatment with 3-MA, a known PI3 kinase inhibitor, only at 32 h and was ineffective if treated later, as high ROS level was already attained. Treatment of F111 and F-HABP07 cells with bafilomycin A1 further indicated an increase in autophagosome formation along with autophagic degradation in HABP1 overexpressed fibroblasts. Comparison between normal fibroblast (F111) and F-HABP07 cells indicate reduced level of polymeric HA, its depolymerization and perturbed HA-HABP1 interaction in F-HABP07. Interestingly, supplementation of polymeric HA, an endogenous ROS scavenger, in the culture medium prompted reduction in number of vacuoles in F-HABP07 along with drop in ROS level, implying that excess ROS generation triggers initiation of autophagic vacuole formation prior to apoptosis due to overexpression of HABP1. Thus, the phenomenon of autophagy takes place prior to apoptosis induction in the HABP1 overexpressing cell line, F-HABP07.
Highlights
The glycosaminoglycan, hyaluronan (HA), an endogenous antioxidant [1,2,3] regulates several cellular pathways via a battery of HA binding proteins or ‘hyaladherins’ [4]
Hyaluronan-binding protein 1 (HABP1) primarily localizes in the mitochondrial matrix but its presence has been reported at the cell surface, nucleus and cytosol suggesting myriad ligand binding capacity attributing to its multifunctional nature [9,16,17,18]
It was observed that HABP1 accumulates in the mitochondria leading to generation of Reactive Oxygen Species (ROS), increased Ca++ influx in mitochondria resulting in membrane potential drop creating mitochondrial dysfunction [16]
Summary
The glycosaminoglycan, hyaluronan (HA), an endogenous antioxidant [1,2,3] regulates several cellular pathways via a battery of HA binding proteins or ‘hyaladherins’ [4]. HA oligomers of 2.5 X 103 have been observed to inhibit anchorage independent growth of tumor cells and to induce apoptosis [7] polymeric HA has been implicated in cellular proliferation [8,9]. HABP1 primarily localizes in the mitochondrial matrix but its presence has been reported at the cell surface, nucleus and cytosol suggesting myriad ligand binding capacity attributing to its multifunctional nature [9,16,17,18]. HepG2 cells, containing an endogenously high level of an array of antioxidant enzymes stably overexpressing HABP1 do not lead to ROS generation, cellular stress and apoptosis rather are more proliferative in nature with increased polymeric HA than their parent cell line [9]
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