Browsing by Author "Hassan, Iraj"

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    Defective cancellous bone structure and abnormal response to PTH in cortical bone of mice lacking Cx43 cytoplasmic C-terminus domain
    (Elsevier, 2015-12) Pacheco-Costa, Rafael; Davis, Hannah M.; Sorenson, Chad; Hon, Mary C.; Hassan, Iraj; Reginato, Rejane D.; Allen, Matthew R.; Bellido, Teresita; Plotkin, Lilian I.; Department of Anatomy & Cell Biology, IU School of Medicine
    Connexin 43 (Cx43) forms gap junction channels and hemichannels that allow the communication among osteocytes, osteoblasts, and osteoclasts. Cx43 carboxy-terminal (CT) domain regulates channel opening and intracellular signaling by acting as a scaffold for structural and signaling proteins. To determine the role of Cx43 CT domain in bone, mice in which one allele of full length Cx43 was replaced by a mutant lacking the CT domain (Cx43(ΔCT/fl)) were studied. Cx43(ΔCT/fl) mice exhibit lower cancellous bone volume but higher cortical thickness than Cx43(fl/fl) controls, indicating that the CT domain is involved in normal cancellous bone gain but opposes cortical bone acquisition. Further, Cx43(ΔCT) is able to exert the functions of full length osteocytic Cx43 on cortical bone geometry and mechanical properties, demonstrating that domains other than the CT are responsible for Cx43 function in cortical bone. In addition, parathyroid hormone (PTH) failed to increase endocortical bone formation or energy to failure, a mechanical property that indicates resistance to fracture, in cortical bone in Cx43(ΔCT) mice with or without osteocytic full length Cx43. On the other hand, bone mass and bone formation markers were increased by the hormone in all mouse models, regardless of whether full length or Cx43(ΔCT) were or not expressed. We conclude that Cx43 CT domain is involved in proper bone acquisition; and that Cx43 expression in osteocytes is dispensable for some but not all PTH anabolic actions.
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    High Bone Mass in Mice Lacking Cx37 Due to Defective Osteoclast Differentiation
    (American Society for Biochemistry and Molecular Biology, 2014-02) Pacheco-Costa, Rafael; Hassan, Iraj; Reginato, Rejane D.; Davis, Hannah M.; Bruzzaniti, Angela; Allen, Matthew R.; Plotkin, Lilian I.; Department of Anatomy & Cell Biology, School of Medicine
    Connexin (Cx) proteins are essential for cell differentiation, function and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts and osteocytes. Mice with global deletion of Cx37 (Cx37-/-) exhibit higher BMD, cancellous bone volume, and mechanical strength compared to wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37-/- mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37-/- mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37+/+ littermates. sRANKL/M-CSF treatment of non-adherent Cx37-/- bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared to Cx37+/+ cell cultures. Further, Cx37-/- osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, MMP9, NFATc1, DCSTAMP, ATP6v0d1 and CD44, markers of osteoclast number, fusion or activity, is lower in Cx37-/- osteoclasts compared to controls. In addition, non-adherent bone marrow cells from Cx37-/- mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo.
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    Investigation of the Mechanism of the Gene Regulation of OPG (Osteoprotegerin) by Cx43
    (Office of the Vice Chancellor for Research, 2013-04-05) Hassan, Iraj; Pacheco-Costa, Rafael; Plotkin, Lillian I.
    The main objective is to determine whether the gene regulation of OPG, osteoprotegerin, by Cx43, is at the promoter level. In a recent project, it was found that deletion in Cx43 from osteocytes resulted in a decreased OPG expression. Furthermore, it was found that deletion of Cx43 from osteocytes resulted in enhanced osteoclast differentiation. Osteoprotegerin (OPG), an osteoblast-secreted decoy receptor that modulates osteoclast formation could be directly controlled by Cx43 at the promoter binding sites of p53 and Sp1. Cx43 and OPG in turn are widely up regulated by Wnt, lipid-modified signaling proteins that influence cell proliferation, differentiation, and survival. The activation of Wnt signaling results in the binding of the transcription factor Tcf in gene promoters, which leads to increased gene expression. The investigation was carried out using reporter constructs in which the activation of the promoter resulted in the transcription of the enzyme luciferase. Luciferase activity, in turn, can be measured using a commercially available substrate that emits luminescence when luciferase is present. OPG-Luc and Tcf-Luc were grown in E. coli and purified using a kit from Qiagen. Transfection of OPG 1 and Tcf-Luc reporter constructs on MLO-Y4 osteocyte cells deleted for Cx43 (Cx43shRNA) and Cx37 (Cx37shRNA) was conducted after seeding of the cells a day in advance. For each cell line, regular and Lithium Chloride (to mimic the effects of Wnt) induced medium was used, and cells were cultured for 24h. From the assay, it was deemed that luciferase activity was higher in Wnt induced cells. OPG is a target of Wnt signaling downstream of the transcription factor Tcf. We therefore also measure Tcf-mediated transcription using a Tcf-luciferase construct. Expression of OPG-Luc and Tcf-Luc was higher in cell lines that are not silenced for Cx43 and Cx37. According to ANOVA test, the results did reach statistical significance. However, future trials will be conducted to mimic the results.
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    Tumor Necrosis Factor Alpha (TNF-α) Disrupts Kir4.1 Channel Expression Resulting in Müller Cell Dysfunction in the RetinaDiurnal Rhythm of Kir4.1 in the Retina
    (ARVO, 2017-05-01) Hassan, Iraj; Luo, Qianyi; Majumdar, Sreeparna; Dominguez, James M.; Busik, Julia V.; Bhatwadekar, Ashay D.; Department of Ophthalmology, IU School of Medicine
    Purpose: Diabetic patients often are affected by vision problems. We previously identified diabetic retinopathy (DR) as a disease of clock gene dysregulation. TNF-α, a proinflammatory cytokine, is known to be elevated in DR. Müller cells maintain retinal water homeostasis and K+ concentration via Kir4.1 channels. Notably, Kir4.1 expression is reduced in diabetes; however, the interplay of TNF-α, Kir4.1, and clock genes in Müller cells remains unknown. We hypothesize that the Kir4.1 in Müller cells is under clock regulation, and increase in TNF-α is detrimental to Kir4.1. Methods: Long-Evans rats were made diabetic using streptozotocin (STZ). Retinal Kir4.1 expression was determined at different time intervals. Rat Müller (rMC-1) cells were transfected with siRNA for Per2 or Bmal1 and in parallel treated with TNF-α (5–5000 pM) to determine Kir4.1 expression. Results: Kir4.1 expression exhibited a diurnal rhythm in the retina; however, with STZ-induced diabetes, Kir4.1 was reduced overall. Kir4.1 rhythm was maintained in vitro in clock synchronized rMC-1 cells. Clock gene siRNA-treated rMC-1 exhibited a decrease in Kir4.1 expression. TNF-α treatment of rMCs lead to a profound decrease in Kir4.1 due to reduced colocalization of Kir4.1 channels with synapse-associated protein (SAP97) and disorganization of the actin cytoskeleton. Conclusions: Our findings demonstrate that Kir4.1 channels possess a diurnal rhythm, and this rhythm is dampened with diabetes, thereby suggesting that the increase in TNF-α is detrimental to normal Kir4.1 rhythm and expression.