IU Indianapolis ScholarWorks :: Browsing by Subject "Cell biology "

Browsing by Subject "Cell biology"

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A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle
(Elsevier, 2023-03-31) Wang, Ruizhong; Kumar, Brijesh; Bhat-Nakshatri, Poornima; Khatpe, Aditi S.; Murphy, Michael P.; Wanczyk, Kristen E.; Simpson, Edward; Chen, Duojiao; Gao, Hongyu; Liu, Yunlong; Doud, Emma H.; Mosley, Amber L.; Nakshatri, Harikrishna; Surgery, School of Medicine
Skeletal muscle dysfunction or reprogramming due to the effects of the cancer secretome is observed in multiple malignancies. Although mouse models are routinely used to study skeletal muscle defects in cancer, because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required. Here, we establish simplified multiple skeletal muscle stem cell lines (hMuSCs), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with the transition of hMuSCs to myotubes. Cancer secretome accelerated stem to myotube differentiation, altered the alternative splicing machinery and increased inflammatory, glucocorticoid receptor, and wound healing pathways in hMuSCs. Additionally, cancer secretome reduced metabolic and survival pathway associated miR-486, AKT, and p53 signaling in hMuSCs. hMuSCs underwent myotube differentiation when engrafted into NSG mice and thus providing a humanized in vivo skeletal muscle model system to study cancer cachexia.
A remarkable adaptive paradigm of heart performance and protection emerges in response to marked cardiac-specific overexpression of ADCY8
(eLife Sciences, 2022-12-14) Tarasov, Kirill V.; Chakir, Khalid; Riordon, Daniel R.; Lyashkov, Alexey E.; Ahmet, Ismayil; Perino, Maria Grazia; Silvester, Allwin Jennifa; Zhang, Jing; Wang, Mingyi; Lukyanenko, Yevgeniya O.; Qu, Jia-Hua; Barrera, Miguel Calvo-Rubio; Juhaszova, Magdalena; Tarasova, Yelena S.; Ziman, Bruce; Telljohann, Richard; Kumar, Vikas; Ranek, Mark; Lammons, John; Bychkov, Rostislav; de Cabo, Rafael; Jun, Seungho; Keceli, Gizem; Gupta, Ashish; Yang, Dongmei; Aon, Miguel A.; Adamo, Luigi; Morrell, Christopher H.; Otu, Walter; Carroll, Cameron; Chambers, Shane; Paolocci, Nazareno; Huynh, Thanh; Pacak, Karel; Weiss, Robert; Field, Loren; Sollott, Steven J.; Lakatta, Edward G.; Medicine, School of Medicine
Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TGAC8, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TGAC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TGAC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TGAC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.
Acquisition, processing, and single-cell analysis of normal human breast tissues from a biobank
(Cell Press, 2021-12-16) Bhat-Nakshatri, Poornima; Marino, Natascia; Gao, Hongyu; Liu, Yunlong; Storniolo, Anna Maria; Nakshatri, Harikrishna; Surgery, School of Medicine
The Komen Tissue Bank is the only biorepository in the world for normal breast tissues from women. Below we report the acquisition and processing of breast tissue from volunteer donors and describe an experimental and analysis pipeline to generate a single-cell atlas. This atlas is based on single-cell RNA-seq and is useful to derive breast epithelial cell subcluster-specific gene expression signatures, which can be applied to breast cancer gene expression data to identify putative cell-of-origin. For complete details on the use and execution of this protocol, please refer to Bhat-Nakshatri et al. (2021).
Cardiomyocyte microRNA-150 confers cardiac protection and directly represses proapoptotic small proline–rich protein 1A
(American Society for Clinical Investigation, 2021-09-22) Aonuma, Tatsuya; Moukette, Bruno; Kawaguchi, Satoshi; Barupala, Nipuni P.; Sepúlveda, Marisa N.; Corr, Christopher; Tang, Yaoliang; Liangpunsakul, Suthat; Payne, R. Mark; Willis, Monte S.; Kim, Il-man; Anatomy, Cell Biology and Physiology, School of Medicine
MicroRNA-150 (miR-150) is downregulated in patients with multiple cardiovascular diseases and in diverse mouse models of heart failure (HF). miR-150 is significantly associated with HF severity and outcome in humans. We previously reported that miR-150 is activated by β-blocker carvedilol (Carv) and plays a protective role in the heart using a systemic miR-150 KO mouse model. However, mechanisms that regulate cell-specific miR-150 expression and function in HF are unknown. Here, we demonstrate that potentially novel conditional cardiomyocyte–specific (CM-specific) miR-150 KO (miR-150 cKO) in mice worsens maladaptive cardiac remodeling after myocardial infarction (MI). Genome-wide transcriptomic analysis in miR-150 cKO mouse hearts identifies small proline–rich protein 1a (Sprr1a) as a potentially novel target of miR-150. Our studies further reveal that Sprr1a expression is upregulated in CMs isolated from ischemic myocardium and subjected to simulated ischemia/reperfusion, while its expression is downregulated in hearts and CMs by Carv. We also show that left ventricular SPRR1A is upregulated in patients with HF and that Sprr1a knockdown in mice prevents maladaptive post-MI remodeling. Lastly, protective roles of CM miR-150 are, in part, attributed to the direct and functional repression of proapoptotic Sprr1a. Our findings suggest a crucial role for the miR-150/SPRR1A axis in regulating CM function post-MI.
Cell non-autonomous requirement of p75 in the development of geniculate oral sensory neurons
(Springer Nature, 2020-12-17) Tang, Tao; Donnelly, Christopher R.; Shah, Amol A.; Bradley, Robert M.; Mistretta, Charlotte M.; Pierchala, Brian A.; Anatomy and Cell Biology, School of Medicine
During development of the peripheral taste system, oral sensory neurons of the geniculate ganglion project via the chorda tympani nerve to innervate taste buds in fungiform papillae. Germline deletion of the p75 neurotrophin receptor causes dramatic axon guidance and branching deficits, leading to a loss of geniculate neurons. To determine whether the developmental functions of p75 in geniculate neurons are cell autonomous, we deleted p75 specifically in Phox2b + oral sensory neurons (Phox2b-Cre; p75fx/fx) or in neural crest-derived cells (P0-Cre; p75fx/fx) and examined geniculate neuron development. In germline p75−/− mice half of all geniculate neurons were lost. The proportion of Phox2b + neurons, as compared to Phox2b-pinna-projecting neurons, was not altered, indicating that both populations were affected similarly. Chorda tympani nerve recordings demonstrated that p75−/− mice exhibit profound deficits in responses to taste and tactile stimuli. In contrast to p75−/− mice, there was no loss of geniculate neurons in either Phox2b-Cre; p75fx/fx or P0-Cre; p75fx/fx mice. Electrophysiological analyses demonstrated that Phox2b-Cre; p75fx/fx mice had normal taste and oral tactile responses. There was a modest but significant loss of fungiform taste buds in Phox2b-Cre; p75fx/fx mice, although there was not a loss of chemosensory innervation of the remaining fungiform taste buds. Overall, these data suggest that the developmental functions of p75 are largely cell non-autonomous and require p75 expression in other cell types of the chorda tympani circuit.
Cilia-associated wound repair mediated by IFT88 in retinal pigment epithelium
(Springer Nature, 2023-05-21) Ning, Ke; Bhuckory, Mohajeet B.; Lo, Chien‑Hui; Sendayen, Brent E.; Kowal, Tia J.; Chen, Ming; Bansal, Ruchi; Chang, Kun‑Che; Vollrath, Douglas; Berbari, Nicolas F.; Mahajan, Vinit B.; Hu, Yang; Sun, Yang; Biology, School of Science
Primary cilia are conserved organelles that integrate extracellular cues into intracellular signals and are critical for diverse processes, including cellular development and repair responses. Deficits in ciliary function cause multisystemic human diseases known as ciliopathies. In the eye, atrophy of the retinal pigment epithelium (RPE) is a common feature of many ciliopathies. However, the roles of RPE cilia in vivo remain poorly understood. In this study, we first found that mouse RPE cells only transiently form primary cilia. We then examined the RPE in the mouse model of Bardet-Biedl Syndrome 4 (BBS4), a ciliopathy associated with retinal degeneration in humans, and found that ciliation in BBS4 mutant RPE cells is disrupted early during development. Next, using a laser-induced injury model in vivo, we found that primary cilia in RPE reassemble in response to laser injury during RPE wound healing and then rapidly disassemble after the repair is completed. Finally, we demonstrated that RPE-specific depletion of primary cilia in a conditional mouse model of cilia loss promoted wound healing and enhanced cell proliferation. In summary, our data suggest that RPE cilia contribute to both retinal development and repair and provide insights into potential therapeutic targets for more common RPE degenerative diseases.
Collagenase-based wound debridement agent induces extracellular matrix supporting phenotype in macrophages
(Springer Nature, 2024-02-08) Banerjee, Pradipta; Das, Amitava; Singh, Kanhaiya; Khanna, Savita; Sen, Chandan K.; Roy, Sashwati; Surgery, School of Medicine
Macrophages assume diverse phenotypes and functions in response to cues from the microenvironment. Earlier we reported an anti-inflammatory effect of Collagenase Santyl® Ointment (CSO) and the active constituent of CSO (CS-API) on wound macrophages in resolving wound inflammation indicating roles beyond debridement in wound healing. Building upon our prior finding, this study aimed to understand the phenotypes and subsets of macrophages following treatment with CS-API. scRNA-sequencing was performed on human blood monocyte-derived macrophages (MDM) following treatment with CS-API for 24 h. Unbiased data analysis resulted in the identification of discrete macrophage subsets based on their gene expression profiles. Following CS-API treatment, clusters 3 and 4 displayed enrichment of macrophages with high expression of genes supporting extracellular matrix (ECM) function. IPA analysis identified the TGFβ-1 pathway as a key hub for the CS-API-mediated ECM-supportive phenotype of macrophages. Earlier we reported the physiological conversion of wound-site macrophages to fibroblasts in granulation tissue and impairment of such response in diabetic wounds, leading to compromised ECM and tensile strength. The findings that CSO can augment the physiological conversion of macrophages to fibroblast-like cells carry significant clinical implications. This existing clinical intervention, already employed for wound care, can be readily repurposed to improve the ECM response in chronic wounds.
Death effector domain-containing protein induces vulnerability to cell cycle inhibition in triple-negative breast cancer
(Springer Nature, 2019-06-28) Ni, Yingjia; Schmidt, Keon R.; Werner, Barnes A.; Koenig, Jenna K.; Guldner, Ian H.; Schnepp, Patricia M.; Tan, Xuejuan; Jiang, Lan; Host, Misha; Sun, Longhua; Howe, Erin N.; Wu, Junmin; Littlepage, Laurie E.; Nakshatri, Harikrishna; Zhang, Siyuan; Surgery, IU School of Medicine
Lacking targetable molecular drivers, triple-negative breast cancer (TNBC) is the most clinically challenging subtype of breast cancer. In this study, we reveal that Death Effector Domain-containing DNA-binding protein (DEDD), which is overexpressed in > 60% of TNBCs, drives a mitogen-independent G1/S cell cycle transition through cytoplasm localization. The gain of cytosolic DEDD enhances cyclin D1 expression by interacting with heat shock 71 kDa protein 8 (HSC70). Concurrently, DEDD interacts with Rb family proteins and promotes their proteasome-mediated degradation. DEDD overexpression renders TNBCs vulnerable to cell cycle inhibition. Patients with TNBC have been excluded from CDK 4/6 inhibitor clinical trials due to the perceived high frequency of Rb-loss in TNBCs. Interestingly, our study demonstrated that, irrespective of Rb status, TNBCs with DEDD overexpression exhibit a DEDD-dependent vulnerability to combinatorial treatment with CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo. Thus, our study provided a rationale for the clinical application of CDK4/6 inhibitor combinatorial regimens for patients with TNBC.
Determinants shaping the nanoscale architecture of the mouse rod outer segment
(eLife Sciences, 2021-12-21) Pöge, Matthias; Mahamid, Julia; Imanishi, Sanae S.; Plitzko, Jürgen M.; Palczewski, Krzysztof; Baumeister, Wolfgang; Ophthalmology, School of Medicine
The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations have been reported to compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkably precise arrangement of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data confirm the existence of two previously observed molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further provide evidence that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) oligomers. We suggest that together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Our Cryo-ET data provide novel quantitative and structural information on the molecular architecture in ROS and substantiate previous results on proposed mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.
GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis
(eLife Sciences, 2022-09-15) Cordova, Ricardo A.; Misra, Jagannath; Amin, Parth H.; Klunk, Anglea J.; Damayanti, Nur P.; Carlson, Kenneth R.; Elmendorf, Andrew J.; Kim, Hyeong-Geug; Mirek, Emily T.; Elzey, Bennet D.; Miller, Marcus J.; Dong, X. Charlie; Cheng, Liang; Anthony, Tracy G.; Pili, Roberto; Wek, Ronald C.; Staschke, Kirk A.; Biochemistry and Molecular Biology, School of Medicine
A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier (SLC) genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels. Addition of essential amino acids or expression of 4F2 (SLC3A2) partially restored growth following loss of GCN2, suggesting that GCN2 targeting of SLC transporters is required for amino acid homeostasis needed to sustain tumor growth. A small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castration-resistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa.

Browsing by Subject "Cell biology"

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