Cell biology
Abstract
Lynch syndrome (LS), caused by inherited mutations in DNA mismatch repair genes including MSH2, carries a 60% lifetime risk of developing endometrial cancer (EC). Beyond hypermutability, mechanisms driving LS-associated EC remain unclear. We investigated MSH2 loss in EC pathogenesis using a mouse model (PR-Cre Msh2LoxP/LoxP, abbreviated Msh2KO), primary cell lines, human tissues, and human EC cells with isogenic MSH2 knockdown. By eight months, 58% of Msh2KO mice developed endometrial atypical hyperplasia (AH), a precancerous lesion. At 12-16 months, 47% of Msh2KO mice exhibited either AH or ECs with histologic similarities to human LS-ECs. Transcriptomic profiling of EC from Msh2KO mice revealed mitochondrial dysfunction-related pathway alterations. Subsequent studies in vitro and in vivo revealed mitochondrial dysfunction based upon two mechanisms: mitochondrial content reduction and structural disruptions in retained mitochondria. Human LS-ECs also exhibited mitochondrial content reduction compared to non-LS-ECs. Functional studies demonstrated metabolic reprogramming of MSH2-deficient EC cells, including reduced oxidative phosphorylation and increased susceptibility to glycolysis suppression. These findings identified mitochondrial dysfunction and metabolic disruption as consequences of MSH2 deficiency in EC. Mitochondrial and metabolic aberrations should be evaluated as biomarkers for endometrial carcinogenesis or risk stratification and represent potential targets for cancer interception in women with LS.
Authors
Mikayla Borthwick Bowen, Brenda Melendez, Qian Zhang, Diana Moreno, Leah Peralta, Wai-Kin Chan, Collene Jeter, Lin Tan, M. Anna Zal, Philip L. Lorenzi, Kenneth Dunner Jr., Richard K. Yang, Russell R. Broaddus, Joseph Celestino, Nisha Gokul, Elizabeth Whitley, Deena M. Scoville, Tae Hoon KIM, Jae-Wook Jeong, Rosemarie Schmandt, Karen Lu, Hyun-Eui Kim, Melinda S. Yates
Abstract
The omentum is the primary site of metastasis for ovarian cancer (OC). Interactions between cancer cells and adipocytes drive an invasive and pro-metastatic phenotype. Here we studied cancer cell-adipocyte crosstalk by using a direct co-culture model with immortalized human visceral pre-adipocytes (VNPAD) and OC cells. We demonstrate increased proliferation, invasiveness, and resistance to cisplatin of co-cultured compared to mono-cultured OC cells. RNA-sequencing of OC cells from co-culture vs. mono-culture revealed significant transcriptomic changes, identifying over 200 differentially expressed genes (DEGs) common to OVCAR5 and OVCAR8 cell lines. Enriched pathways included PI3K/AKT and Complement activation. Lipid transfer into OC cells from adipocytes induced upregulation of complement C3 and C5 proteins. Inhibiting C3 or C5 reversed the invasive phenotype and C3 knockdown reduced tumor progression in-vivo. Increased C3 expression was observed in omental implants compared to primary ovarian tumors and C3 secretion was higher in OC ascites from high BMI vs. low BMI patients. C3 upregulation in OC cells involved activation of ATF4-mediated integrated stress response (ISR). Overall, adipocyte-cancer cell interactions promote invasiveness and tumorigenesis via lipid transfer, activating ISR, and upregulating complement proteins C3 and C5.
Authors
Andres Valdivia, Ana Isac, Horacio Cardenas, Guangyuan Zhao, Yaqi Zhang, Hao Huang, Jian-Jun Wei, Mauricio Cuello-Fredes, Sumie Kato, Fernán Gómez-Valenzuela, Francoise A. Gourronc, Aloysius J. Klingelhutz, Daniela Matei
Abstract
Hypertension and transient increases in blood pressure from extreme exertion are risk factors for aortic dissection in patients with age-related vascular degeneration or inherited connective tissue disorders. Yet, a common experimental model of angiotensin II-induced aortopathy in mice appears independent of high blood pressure as lesions do not occur in response to an alternative vasoconstrictor, norepinephrine, and are not prevented by co-treatment with a vasodilator, hydralazine. We investigated vasoconstrictor administration to adult mice following 1 week of disrupted TGFβ signaling in smooth muscle cells (SMCs). Norepinephrine increased blood pressure and induced aortic dissection by 7 days and even within 30 minutes (as did angiotensin II) that was prevented by hydralazine. Initial medial injury manifested as blood extravasation among SMCs and fibrillar matrix, progressive delamination from accumulation of blood, and stretched or ruptured SMCs with persistent attachments to elastic fibers. Altered regulatory contractile molecule expression was not of pathological importance. Rather, reduced synthesis of extracellular matrix yielded a vulnerable aortic phenotype by decreasing medial collagen, most dynamically basement membrane-associated multiplexin collagen, and impairing cell-matrix adhesion. We conclude that transient and sustained increases in blood pressure can cause dissection in aortas rendered vulnerable by inhibition of TGFβ-driven extracellular matrix production by SMCs.
Authors
Bo Jiang, Pengwei Ren, Changshun He, Mo Wang, Sae-Il Murtada, María Jesús Ruiz-Rodríguez, Yu Chen, Abhay B. Ramachandra, Guangxin Li, Lingfeng Qin, Roland Assi, Martin A. Schwartz, Jay D. Humphrey, George Tellides
Abstract
Temporomandibular joint (TMJ) osteoarthritis with pain is a highly prevalent disorder affecting patients’ quality of life. A comprehensive understanding of cell type diversity and its dynamics in painful TMJ osteoarthritis (TMJOA) is lacking. Here, we utilized an inflammatory TMJOA mouse model via intra-articular injection of CFA. TMJOA mice exhibited cartilage remodeling, bone loss, synovitis, increased osteoarthritis (OA) score, and orofacial pain, recapitulating hallmark symptoms in patients. Single-cell transcriptomic profiling of the TMJ was performed in conjunction with mouse genetic labeling, tissue clearing, light sheet and confocal 3D imaging, multiplex RNAscope, and immunodetection. We visualized, reconstructed, and analyzed the distribution and density of nociceptive innervation of TMJ at single-axon levels. We systematically mapped the heterogeneity and anatomical position of blood endothelial cells, synovial fibroblasts, and immune cells, including Cx3cr1-positive barrier macrophages. Importantly, TMJOA mice exhibited enhanced neurovascular coupling, sublining fibroblast hyperplasia, inflammatory immune cell expansion, disrupted signaling-dependent cell-cell interaction, and a breakdown of the sandwich-like organization consisting of synovial barrier macrophages and fibroblasts. By utilizing a mouse model with combined TMJ pain history and OA, we reveal the cellular diversity, anatomical structure, and cell dynamics of the TMJ at single-cell resolution, which facilitate our understanding and potential targeting of TMJOA.
Authors
Supawadee Jariyasakulroj, Yang Shu, Ziying Lin, Jingyi Chen, Qing Chang, Pao-Fen Ko, Jian-Fu Chen
Abstract
The proof-of-principle of the therapeutic potential of heat shock protein 47 (HSP47) for diseases characterized by defects in the collagen I synthesis is here proved in osteogenesis imperfecta (OI), a prototype of collagen disorders. Most of the OI mutations delay collagen I chains folding, increasing their exposure to post translational modifications that affect collagen secretion and impact extracellular matrix fibrils assembly. As model, we used primary fibroblasts from OI individuals with defect in the collagen prolyl-3-hydroxylation complex, since are characterized by the synthesis of homogeneously overmodified collagen molecules. We demonstrated that the exogenous recombinant HSP47 (rHSP47) is uptaken by the cells and localizes at the ER exit sites and ER Golgi intermediate compartment. rHSP47 treatment increased collagen secretion, reduced collagen post translational modifications and intracellular collagen retention and ameliorated the general ER proteostasis, leading to improved cellular homeostasis and vitality. These positive changes were also mirrored by an increased collagen content in the OI matrix. A mutation dependent effect was found in fibroblasts from three probands with collagen I mutations, for which rHSP47 was effective only in cells with the most N-term defect. A beneficial effect on bone mineralization was proved in vivo in the zebrafish p3h1-/- OI model.
Authors
Roberta Besio, Nadia Garibaldi, Alessandra Sala, Francesca Tonelli, Carla Aresi, Elisa Maffioli, Claudio Casali, Camilla Torriani, Marco Biggiogera, Simona Villani, Antonio Rossi, Gabriella Tedeschi, Antonella Forlino
Abstract
Lymphangioleiomyomatosis (LAM) is a progressive lung disease with limited treatments, largely due to an incomplete understanding of its pathogenesis. Lymphatic endothelial cells (LECs) invade LAM cell clusters, which include HMB-45-positive epithelioid cells and smooth muscle α-actin-expressing LAM-associated fibroblasts (LAMFs). Recent evidence shows that LAMFs resemble cancer-associated fibroblasts, with LAMF-LEC interactions contributing to disease progression. To explore these mechanisms, we used spatial transcriptomics on LAM lung tissues and identified a gene cluster enriched in kinase signaling pathways linked to myofibroblasts and co-expressed with LEC markers. Kinase arrays revealed elevated PDGFR and FGFR in LAMFs. Using a 3D co-culture spheroid model of primary LAMFs and LECs, we observed increased invasion in LAMF-LEC spheroids compared to non-LAM fibroblasts. Treatment with sorafenib, a multikinase inhibitor, significantly reduced invasion, outperforming Rapamycin. We also confirmed TSC2-deficient renal angiomyolipoma cells (TSC2-null AML) as key VEGF-A secretors, which was suppressed by sorafenib in both TSC2-null AML cells and LAMFs. These findings highlight VEGF-A and bFGF as potential therapeutic targets and suggest multikinase inhibition as a promising strategy for LAM.
Authors
Sinem Koc-Gunel, Emily C. Liu, Lalit K. Gautam, Ben A. Calvert, Shubha Murthy, Noa C. Harriott, Janna C. Nawroth, Beiyun Zhou, Vera P. Krymskaya, Amy L. Ryan
Abstract
Renal osteodystrophy is commonly seen in patients with chronic kidney disease (CKD) due to disrupted mineral homeostasis. Given the impaired renal function in these patients, common anti-resorptive agents, including bisphosphonates, must be used with caution or even contraindicated. Therefore, an alternative therapy without renal burden to combat renal osteodystrophy is urgently needed. Here, we report that clinically relevant aerobic exercise significantly prevents high-turnover renal osteodystrophy in CKD mouse and patients without compromising renal function. Mechanistically, 4-week aerobic exercise in CKD mice increased expression of skeletal muscle PPARγ coactivator-1α (PGC-1α) and circulating irisin. Both exercise and irisin administration significantly activated osteoblasts, but not osteoclasts, via integrin αvβ5, thereby conferring bone quality benefits. Removal of irisin-influenced thermogenic adipose tissues or genetic ablation of uncoupling protein 1 did not alter the irisin-conferred anti-osteodystrophy effect. Importantly, in a pilot clinical study, 12-week aerobic exercise in patients with high-grade CKD significantly increased circulating irisin and prevented osteodystrophy progression, without detectable renal burden. The combination of irisin and current anti-resorptive agents effectively rescued renal osteodystrophy in mice. Our work provides mechanistic insights into the role of exercise and irisin in renal osteodystrophy, and highlights a clinically relevant, low-cost, kidney-friendly therapy for patients with this devastating disease.
Authors
Meng Wu, Huilan Li, Xiaoting Sun, Rongrong Zhong, Linli Cai, Ruibo Chen, Madiya Madeniyet, Kana Ren, Zhen Peng, Yujie Yang, Weiqin Chen, Yanling Tu, Miaoxin Lai, Jinxiu Deng, Yuting Wu, Shumin Zhao, Qingyan Ruan, Mei Rao, Sisi Xie, Ying Ye, Jianxin Wan
Abstract
Skeletal muscle regeneration in adults is predominantly driven by satellite cells. Loss of satellite cell pool and function leads to skeletal muscle wasting in many conditions and disease states. Here, we demonstrate that the levels of fibroblast growth factor-inducible 14 (Fn14) were increased in satellite cells after muscle injury. Conditional ablation of Fn14 in Pax7-expressing satellite cells drastically reduced their expansion and skeletal muscle regeneration following injury. Fn14 was required for satellite cell self-renewal and proliferation as well as to prevent precocious differentiation. Targeted deletion of Fn14 inhibited Notch signaling but led to the spurious activation of STAT3 signaling in regenerating skeletal muscle and in cultured muscle progenitor cells. Silencing of STAT3 improved proliferation and inhibited premature differentiation of Fn14-deficient satellite cells. Furthermore, conditional ablation of Fn14 in satellite cells exacerbated myopathy in the mdx mouse model of Duchenne muscular dystrophy (DMD) whereas its overexpression improved the engraftment of exogenous muscle progenitor cells into the dystrophic muscle of mdx mice. Altogether, our study highlights the crucial role of Fn14 in the regulation of satellite cell fate and function and suggests that Fn14 can be a potential molecular target to improve muscle regeneration in muscular disorders.
Authors
Meiricris Tomaz da Silva, Aniket S. Joshi, Ashok Kumar
Abstract
Surgery of the tracheobronchial tree carries high morbidity, with over half of the complications occurring at the anastomosis. Although fibroblasts are crucial in airway wound healing, the underlying cellular and molecular mechanisms in airway reconstruction remain unknown. We hypothesized that airway reconstruction initiates a surgery-induced stress (SIS) response, altering fibroblast communication within airway tissues. Using single-cell RNAseq, we analyzed native and reconstructed airways and identified five fibroblast subpopulations, each with distinct spatial distributions across anastomotic, submucosal, perichondrial, and paratracheal areas. During homeostasis, Adventitial and Airway fibroblasts (Adventitial Fb and Airway Fb, respectively) maintained tissue structure and created cellular niches by regulating ECM turnover. Under SIS, Perichondrial fibroblasts (PC-Fb) exhibited chondroprogenitor-like gene signatures, and Immune-recruiting fibroblasts (IR-Fb) facilitated cell infiltration. Cthrc1 activated fibroblasts (Cthrc1+ Fb), mainly derived from Adventitial Fb, primarily contributed to fibrotic scar formation and collagen production, mediated by TGFβ. Furthermore, repeated SIS created an imbalance in fibroblast states favoring emergence of CTHRC1+ Fb and leading to impaired fibroblasts-basal cell crosstalk. Collectively, these data identify PC, IR, and Cthrc1+ Fb as a signaling hub, with SIS emerging as a mechanism initiating airway remodeling after reconstruction that, if not controlled, may lead to complications such as stenosis or anastomotic breakdown.
Authors
Jazmin Calyeca, Zakarie Hussein, Zheng Hong Tan, Lumei Liu, Sayali Dharmadhikari, Kimberly M. Shontz, Tatyana A. Vetter, Christopher K. Breuer, Susan D. Reynolds, Tendy Chiang
Abstract
The oral mucosa undergoes daily insults, and stem cells in the epithelial basal cell layer regenerate gingiva tissue to maintain oral health. The Iroquois Homeobox 1 (IRX1) protein is expressed in the stem cell niches in human/mouse oral epithelium and mesenchyme under homeostasis. We found that Irx1+/– heterozygous (Het) mice have delayed wound closure, delayed morphological changes of regenerated epithelium, and defective keratinocyte proliferation and differentiation during wound healing. RNA-Seq analyses between WT and Irx1+/– mice at 3 days postinjury (dpi) found impaired epithelial migration and decreased keratinocyte-related genes upon injury. IRX1-expressing cells are found in the gingival epithelial basal cell layer, a stem cell niche for gingival maintenance. IRX1-expressing cells are also found in cell niches in the underlying stroma. IRX1 activates SOX9 in the transient amplifying layer to increase cell proliferation, and EGF signaling is activated to induce cell migration. Krt14CreERT lineage tracing experiments reveal defects in the stratification of the Irx1+/– HET mouse oral epithelium. IRX1 is primed at the base of the gingiva in the basal cell layer of the oral epithelium, facilitating rapid and scarless wound healing through activating SOX9 and the EGF signaling pathway.
Authors
Dan Su, Tadkamol Krongbaramee, Samuel Swearson, Yan Sweat, Mason Sweat, Fan Shao, Steven Eliason, Brad A. Amendt
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