Alteration of hedgehog signaling by chronic exposure to different pesticide formulations and unveiling the regenerative potential of recombinant sonic hedgehog in mouse model of bone marrow aplasia

Abstract

Chronic pesticide exposure-induced downregulation of hedgehog signaling and its subsequent degenerative effects on the mammalian hematopoietic system have not been investigated yet. However a number of concurrent studies have pointed out the positive correlation between chronic pesticide exposure induced bone marrow failure and immune suppression. Here, we have given an emphasis on the recapitulation of human marrow aplasia like condition in mice by chronic mixed pesticide exposures and simultaneously unravel the role of individual pesticides in the said event. Unlike the effect of mixed pesticide, individual pesticides differentially alter the hedgehog signaling in the bone marrow primitive hematopoietic compartment (Sca1 + compartment) and stromal compartment. Individually, hexaconazole disrupted hematopoietic as well as stromal hedgehog signaling activation through inhibiting SMO and facilitating PKC δ expression. On contrary, both chlorpyriphos and cypermethrin increased the sequestration and degradation of GLI1 by upregulating SU(FU) and βTrCP, respectively. However, cypermethrin-mediated inhibition of hedgehog signaling has partly shown to be circumvented by non-canonical activation of GLI1. Finally, we have tested the regenerative response of sonic hedgehog and shown that in vitro supplemented recombinant SHH protein augmented clonogenic stromal progenitors (CFU-F) as well as primitive multipotent hematopoietic clones including CFU-GEMM and CFU-GM of mixed pesticide-induced aplastic marrow. It is an indication of the marrow regeneration. Finally, our findings provide a gripping evidence that downregulated hedgehog signaling contribute to pesticide-mediated bone marrow aplasia but it could be recovered by proper supplementation of recombinant SHH along with hematopoietic base cocktail. Furthermore, SU(FU) and GLI1 can be exploited as future theradiagnostic markers for early marrow aplasia diagnosis.

Aberrant Wnt Signaling Pathway in the Hematopoietic Stem/Progenitor Compartment in Experimental Leukemic Animal

“…LT‐BMC is a powerful technique to study BM microenvironmental scenario in vitro by utilizing the ability of a specific marrow cell population to form adherent stromal cell layer (representative of microenvironmental stromal cells in vivo ) under defined culture condition (Ramakrishnan et al 2013; Chaklader and Law 2015). Comparative LT‐BMC of control and leukemic BM derived cells showed different scenario.…”

“…LT-BMC was used to grow BM stromal cells. Stromal cells were stained with anti-Wnt3a and anti-Wnt5a antibody (1:300 dilution) overnight at 4oC according to the protocol of (Chaklader and Law 2015). Cells were washed and stained the following day for 2 h with HRP tagged secondary antibody (1:600 dilution) (Invitrogen).…”

Network-Based Method for Identifying Co-Regeneration Genes in Bone, Dentin, Nerve and Vessel Tissues

“…GLI1 (ENSP00000228682) encoding a member of the Kruppel family of zinc finger proteins, has also been inferred as a bone-nerve co-regeneration gene. According to recent publications, GLI1 has been confirmed to regulate the regenerative capacity of osseous tissues by participating in hedgehog signaling pathway as we have analyzed above [ 58 , 59 ]. As for its potential role in nerve system, considering that hedgehog signaling pathway has also been confirmed to participate in peripheral nerve regeneration [ 60 ], as one unique component of such pathway, GLI1 has also been reported to be downregulated during such regenerative processes [ 61 ].…”

Morphogen signaling by Wnt/β‐catenin pathway and microenvironmental alteration in the bone marrow of agricultural pesticide exposure‐induced experimental aplastic anemia

“…(a) The experimental group of mice was exposed to 5% aqueous pesticide formulation, via inhalation and dermal route, for 6 hour per day 5 days in a week up to 90 days (LD50 of 10% aqueous solution was found to be 20 days as per our previous experiment). [42,43,52] To mimic the practical field scenario, we used common agricultural pesticides such as cypermethrin (Sigma-Aldrich), hexaconazole (Sigma-Aldrich), and chlorpyriphos (Sigma-Aldrich) for pesticide mixture preparation to induce BM aplasia. (b) The control group of mice was exposed to aqueous saline solution without any trace of pesticides for the same duration as experimental group.…”

“…It is an important tool in cytology for studying BM stromal cells in vitro by utilizing the ability of these cells to form an adherent layer under the defined culture condition. [52][53][54] Stromal cells were grown from both control and pesticide-exposed marrow-derived cells. A total of 2 Figure 1F),…”