Kasturi Datta

Plasmodium falciparum Uses gC1qR/HABP1/p32 as a Receptor to Bind to Vascular Endothelium and for Platelet-Mediated Clumping

The ability of Plasmodium falciparum–infected red blood cells (IRBCs) to bind to vascular endothelium, thus enabling sequestration in vital host organs, is an important pathogenic mechanism in malaria. Adhesion of P. falciparum IRBCs to platelets, which results in the formation of IRBC clumps, is another cytoadherence phenomenon that is associated with severe disease. Here, we have used in vitro cytoadherence assays to demonstrate, to our knowledge for the first time, that P. falciparum IRBCs use the 32-kDa human protein gC1qR/HABP1/p32 as a receptor to bind to human brain microvascular endothelial cells. In addition, we show that P. falciparum IRBCs can also bind to gC1qR/HABP1/p32 on platelets to form clumps. Our study has thus identified a novel host receptor that is used for both adhesion to vascular endothelium and platelet-mediated clumping. Given the association of adhesion to vascular endothelium and platelet-mediated clumping with severe disease, adhesion to gC1qR/HABP1/p32 by P. falciparum IRBCs may play an important role in malaria pathogenesis.

Constitutive expression of hyaluronan binding protein 1 (HABP1/p32/gC1qR) in normal fibroblast cells perturbs its growth characteristics and induces apoptosis

Hyaluronan binding protein 1 (HABP1) is a ubiquitously expressed multifunctional phospho-protein that interacts with a wide range of ligands and is implicated in cell signalling. Recently, we have reported that HABP1 is an endogenous substrate for MAP kinase and upon mitogenic stimulation it is translocated to the nucleus in a MAP kinase-dependent manner (Biochem. Biophys. Res. Commun. 291(4) (2002) 829-837). This prompted us to investigate the role of HABP1 in cell growth or otherwise in low MAP kinase background. We demonstrate that HABP1, when overexpressed in normal rat skin fibroblasts, remained in the cytosol, primarily concentrated around the nuclear periphery. However, HABP1 overexpressing cells showed extensive vacuolation and reduced growth rate, which was corrected by frequent medium replenishment. Further investigation revealed that HABP1 overexpressing cells undergo apoptosis, as detected by TUNEL assay, induction of Bax expression, and FACS analysis, and they failed to enter into the S-phase. Periodic medium supplementation prevented these cells from undergoing apoptotic death. We also demonstrate that upon induction of apoptosis in HeLa cells by cisplatin, HABP1 level is upregulated, indicating a correlation between HABP1 and cell death in a normal cellular environment.

Excessive reactive oxygen species induces apoptosis in fibroblasts : Role of mitochondrially accumulated hyaluronic acid binding protein 1 (HABP1/p32/gC1qR)

Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities along with initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca 2+ influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.

Possible role of hyaluronectin on cell adhesion in rat histiocytoma

Distribution of hyaluronectin, a 68-kDa cell surface glycoprotein, has been demonstrated in normal peritoneal, alveolar macrophages as well as in macrophages of the AK-5 tumor cell line. AK-5, a transplantable histiocytic tumor cell line, is a mixture of four different populations and can be grown in both ascites and solid tumors. We are able to demonstrate a differential expression of hyaluronectin on the cell surface of these subpopulations of AK-5 when studied by immunocytochemical staining followed by cytofluorometric analysis. Cell fractions responsible for developing both ascites and solid tumors contain higher amounts of hyaluronectin than fractions which are capable of producing only ascites, suggesting its involvement in solid tumor formation. Furthermore, we established a secretory nature of hyaluronectin as it can be detected in the serum-free medium of AK-5 cells. Since it is localized on the cell surface and secreted into the medium, the cell adhesiveness of hyaluronectin has been examined. Hyaluronectin coating on the plates allowed more cells to attach, which could be specifically blocked by the antibody raised against hyaluronectin, indicating its possible role in cell attachment. The adhesive property of hyaluronectin and its role in tumor formation was further confirmed. The pretreatment of AK-5 cells with hyaluronectin antibody abolished their capacity to grow as solid tumors; however, the cells retained their capacity to grow as ascites tumor. We discuss our observations of hyaluronectin as a cell attachment protein and its specific role on tumor formation.

Hyaluronan-binding protein 1 (HABP1/p32/gC1qR) induces melanoma cell migration and tumor growth by NF-kappa B dependent MMP-2 activation through integrin αvβ3 interaction

Cell migration is the hallmark of cancer regulating anchorage independent growth and invasiveness of tumor cells. Hyaluronan (HA), an ECM polysaccharide is shown to regulate this process. In the present report, we demonstrated, supplementation of purified recombinant hyaluronan binding protein 1(HABP1/p32/gC1qR) from human fibroblast cDNA enhanced migration potential of highly invasive melanoma (B16F10) cells. Exogenous HABP1 adhered to the cell surface transiently and was shown to interact and colocalize with α(v)β(3) integrin, a regulatory molecule of cell migration. In HABP1 treated cells, the phosphorylation of nuclear factor inducing kinase (NIK) and IκBα was observed, followed by nuclear translocation of p65 subunit of NFκB, along with its DNA-binding and transactivation, resulting in upregulation of MT1-MMP expression and finally MMP-2 activation. To substantiate our findings, prior to HABP1 treatment, the expression of NIK was reduced by small interfering RNA mediated knockdown and confirmed the inhibition of nuclear translocation of p65 subunit of NFκB and upregulation of MT1-MMP expression. In addition, the use of curcumin, an anti-cancer drug, or GRGDSP, the blocking peptide along with exogenous HABP1, inhibited such NFκB-dependent pathway, confirming that HABP1-induced cell migration is α(v)β(3) integrin-mediated and downstream signaling by NFκB. Finally, we translated the in vitro data in mice model and observed enhanced tumor growth with higher MT1-MMP expression and MMP-2 activation in the tumors upon injection of HABP1 treated melanoma cells. The treatment of curcumin, the anticancer drug along with HABP1, inhibited the migration, expression of MT1-MMP and activation of MMP-2 and finally tumor growth supports the involvement of HABP1 in tumor formation.

Differential expression of Hyaluronic Acid Binding protein 1 (HABP1)/P32/C1QBP during progression of epidermal carcinoma

Our laboratory has characterized a novel cell surface glycoprotein, Hyaluronic Acid Binding Protein 1 (HABP1), interacting specifically with hyaluronan (HA) and regulating HA-mediated cellular event. The involvement of HA in different stages of carcinoma is well documented. In the present communication, the expression profile of HABP1 was investigated from initiation to progression of epidermal carcinoma in mice, induced by benzo[a]pyrene (B[a]P) exposure. During tumor initiation, HABP1 accumulated in inflammatory subsquamous tissue and with progression, the protein, was also seen to overexpress in papillomatic and acanthotic tissue. With the onset of metastasis, HABP1 overexpression was confined to metastatic islands, while it disappeared gradually from the surrounding mass. Such expression profiles in metastasized tissue were supported by decreased levels of HABP1, both at protein and transcript levels. These observations taken together suggest that the changes in HABP1 level coincide with specific stages of tumor progression, that lead to disruption of its interaction with HA, implying a role in the regulation of tumor metastasis. (Mol Cell Biochem 267: 133–139, 2004)

Multifunctional activities of human fibroblast 34-kDa hyaluronic acid-binding protein.

We have already reported that human fibroblast 34-kDa hyaluronic acid-binding protein (HABP) is identical with P32, the protein co-purified with splicing factor SF-2 [Deb and Datta (1996) J. Biol. Chem. 271, 2206-2212]. Data search further revealed that it has 92% sequence homology with a murine protein YL2 which interacts with HIV1 Rev. In this paper we have successfully demonstrated that HIV1 Rev binds with labeled 34-kDa HABP which can be competed with excess unlabeled HABP, suggesting this protein can be a cellular factor promoting HIV1 Rev to function. Interestingly, the multifunctional nature of HABP has been elucidated as it has 100% homology with another protein gC1q, the complement protein. The distinct non-overlapping binding motifs for HA and gC1q have been identified in the same protein, suggesting that either the protein can function independently or its activity is regulated by ligand binding, wherein its binding to one of the ligands may modulate the receptor activity of the other ligand.

Hyaluronic acid induced hyaluronic acid binding protein phosphorylation and inositol triphosphate formation in lymphocytes

In this report, the role of 34 kDa HA‐binding protein in hyaluronic acid‐induced cellular signalling in lymphocytes has been examined. The binding of 125I‐HA to lymphocytes in vivo was found to be inhibited by pre‐incubation of the cells with anti‐34 kDa HA‐binding protein antibodies, thus confirming 34 kDa HA‐binding protein as the specific HA‐receptor in lymphocytes. This observation was substantiated by anti‐34 kDa HA‐binding protein antibodies immunoblotting and 125I‐HA ligand blotting of lymphocytes cell lysate. The HA‐induced cell aggregation, tyrosine phosphorylation and cytoskeletal protein phosphorylation demonstrate the HA‐induced early cellular signalling events in lymphocytes. Further, to study the involvement of 34 kDa HA‐binding protein in mitogen induced lymphocyte signalling, we studied in vivo phosphorylation and secondary messenger formation. The enhanced 34 kDa HA‐binding protein phosphorylation by HA and the inhibition of cellular aggregation and IP3 formation by anti‐HA‐binding protein antibodies revealed that 34 kDa HA‐binding protein is one of the potential mediators in HA‐induced signal transduction.

Reduction in the level of hyaluronan binding protein 1 (HABP1) is associated with loss of sperm motility

Hyaluronan binding protein 1 (HABP1) was reported to be present on human sperm surface and its involvement in fertilization has already been elucidated (Mol. Repro. Dev. 38 (1994) 69). In the present communication, we report a significant reduction in the level of this protein in sperms from asthenozoospermic and oligozoospermic patients as compared to normozoospermic one. Further evidence of the absence of HABP1 in sperms, having motility <20% is documented, which again is a determining factor for fertilization. HABP1 was quantitatively determined using anti-HABP1 antibody from sperm extracts isolated from semen samples of both the fertile and infertile groups demonstrating low sperm motility. Sperm samples with low motility revealed a significant reduction in the level of HABP1 in immunoblot detection as well as immunolocalization experiment. It suggests that decreased HABP1 level may be associated with low motility of sperms, which in turn might cause infertility in the patient. Thus, the sperm surface HABP1 level can be correlated with the degree of sperm motility, an important criteria for fertilization.

Overexpression of Hyaluronan-binding Protein 1 (HABP1/p32/gC1qR) in HepG2 Cells Leads to Increased Hyaluronan Synthesis and Cell Proliferation by Up-regulation of Cyclin D1 in AKT-dependent Pathway

Background: Hyaluronan (HA) levels regulate cell behavior, tumor invasion, and migration through interactions with hyaladherins. Results: Elevated expression of hyaluronan-binding protein 1 (HABP1) leads to enhanced HA synthesis, HA cable formation, and activation of cell survival pathways in HepG2 cells. Conclusion: Constitutively elevated expression of HABP1 leads to enhanced tumorigenic potential by HA-mediated pathways. Significance: HABP1 modulates cell survival through enhanced HA synthesis. Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased “HA pool,” formation of the “HA cable” structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.