Biology subjects

Robling, A. G.

Publications and source records attributed to Robling, A. G..

9 recordsLinked to original sources

bioRxiv2025

Hepatic Yap1 activates systemic catabolism and muscle loss during organ repair: evidence for a liver-derived common mechanism with cancer cachexia.

Recovery from critical injury concomitant with restoration of functional organ mass invokes a systemic catabolic response leading to muscle and fat loss, known as cachexia. We interrogated this process using mouse models of organ repair, including liver regeneration after hepatectomy, Yap1-mediated hepatomegaly, and pneumonectomy. Both hepatectomy and Yap1 induced systemic catabolism. Muscle and adipose wasting scaled with degree of liver growth, with 10-25% reduction of muscle mass and 50-80% of fat mass. In contrast, non-regenerative lung injury did not induce tissue wasting. Liver growth elevated resting energy expenditure. Tracer studies demonstrated redistribution of muscle-derived cholesterol and amino acids to regenerating liver. Gene expression changes in livers and muscles showed high concordance between the liver growth models and cancer cachexia models, including pancreatic adenocarcinoma cachexia. We propose that cachexia is a normal and essential reparative process in organ repair and regeneration and further, that cancer cachexia is a pathological exacerbation of an adaptive process mediated by activation of Yap1 in the liver.

physiology↗

bioRxiv2023

Gemcitabine plus nab-paclitaxel preserves skeletal and cardiac mass and function in a murine model of pancreatic cancer cachexia

More than 85% of patients with pancreatic ductal adenocarcinoma (PDAC) suffer from cachexia, a debilitating syndrome characterized by the loss of muscle and fat and remains an unmet medical need. While chemotherapy remains an effective treatment option, it can also induce weight and muscle loss in patients with cancer. Gemcitabine combined with nab paclitaxel (GnP) is a first line treatment option for patients with PDAC but GnPs effect on cachexia has not been comprehensively investigated. We interrogated the effects of GnP in a murine model of pancreatic cancer cachexia. Mice were orthotopically implanted with the cachexia inducing pancreatic cell line (KPC) and were administered GnP or vehicle. The controls underwent sham surgery. We defined GnP effects on cachexia and tumor burden by evaluating muscle and cardiac mass and function, fat mass, bone morphometry, and hematology measurements. We completed RNA sequencing and deep proteome profiling in skeletal and cardiac muscle. KPC+GnP reduced tumor burden over 50% and increased survival compared to KPC. KPC vehicle group had more than 15% muscle mass loss and decreased left ventricular mass, this was not present in KPC+GnP when compared to controls. RNA Seq and deep proteomics analyses suggested that muscle and cardiac dysfunction pathways activated in KPC group were either reversed or decreased in KPC+GnP. In all, our data suggests that GnP protects against muscle and cardiac wasting in an experimental model of PDAC cachexia. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/536434v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1d394f7org.highwire.dtl.DTLVardef@1d8fdaforg.highwire.dtl.DTLVardef@f709d4org.highwire.dtl.DTLVardef@a668e0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

cancer biology↗

bioRxiv2022

Single Cell Cortical Bone Transcriptomics Defines Novel Osteolineage Gene Sets Altered in Chronic Kidney Disease

Due to a lack of spatial-temporal resolution at the single cell level, the etiologies of the bone dysfunction caused by diseases such as normal aging, osteoporosis, and the metabolic bone disease associated with chronic kidney disease (CKD) remain largely unknown. To this end, flow cytometry and scRNAseq were performed on long bone cells from Sost-cre/Ai9+ mice, and pure osteolineage transcriptomes were identified, including novel osteocyte-specific gene sets. Clustering analysis isolated osteoblast precursors that expressed Tnc, Mmp13, and Spp1, and a mature osteoblast population defined by Smpd3, Col1a1, and Col11a1. Osteocytes were demarcated by Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, and Phex. We validated our in vivo scRNAseq using integrative in vitro promoter occupancy via ATACseq coupled with transcriptomic analyses of a conditional, temporally differentiated MSC cell line. Further, trajectory analyses predicted osteoblast-to-osteocyte transitions via defined pathways associated with a distinct metabolic shift as determined by single-cell flux estimation analysis (scFEA). Using the adenine mouse model of CKD, at a time point prior to major skeletal alterations, we found that gene expression within all stages of the osteolineage was disturbed. In sum, distinct populations of osteoblasts/osteocytes were defined at the single cell level. Using this roadmap of gene assembly, we demonstrated unrealized molecular defects across multiple bone cell populations in a mouse model of CKD, and our collective results suggest a potentially earlier and more broad bone pathology in this disease than previously recognized.

cell biology↗

bioRxiv2022

Gabapentin Disrupts Binding of Perlecan to the α2δ1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation

Our understanding of how osteocytes, the principal mechanosensors within bone, sense and perceive force remains unclear. Previous work identified "tethering elements" (TEs) spanning the pericellular space of osteocytes and transmitting mechanical information into biochemical signals. While we identified the heparan sulfate proteoglycan perlecan (PLN) as a component of these TEs, PLN must attach to the cell surface to induce biochemical responses. As voltage-sensitive calcium channels (VSCCs) are critical for bone mechanotransduction, we hypothesized that PLN binds the extracellular 2{delta}1 subunit of VSCCs to couple the bone matrix to the osteocyte membrane. Here, we showed co-localization of PLN and 2{delta}1 along osteocyte dendritic processes. Additionally, we quantified the molecular interactions between 2{delta}1 and PLN domains and demonstrated for the first time that 2{delta}1 strongly associates with PLN via its domain III. Furthermore, 2{delta}1 is the binding site for the commonly used pain drug, gabapentin (GBP), which is associated with adverse skeletal effects when used chronically. We found that GBP disrupts PLN::2{delta}1 binding in vitro, and GBP treatment in vivo results in impaired bone mechanosensation. Our work identified a novel mechanosensory complex within osteocytes composed of PLN and 2{delta}1, necessary for bone force transmission and sensitive to the drug GBP. This work provides insights into the mechanisms underlying mechanotransduction and will inform future studies to understand the mechanisms responsible for the negative effects of GBP on bone.

molecular biology↗

bioRxiv2021

Suppression of sost/sclerostin and dikkopf-1 promote intervertebral disc structure in mice

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and characterized by accelerated extracellular matrix breakdown and IVD height loss but there is no approved pharmacological therapeutic. Deletion of Wnt signaling receptor Lrp5 induces IVD degeneration and suggests that Wnt signaling in the IVD may be responsive to inhibition of Wnt signaling inhibitors sost(gene)/sclerostin(protein) or dickkopf-1 (dkk1). Anti-sclerostin antibody (Scl-Ab) is an FDA-approved bone therapeutic that activates Wnt signaling. We (1) determined if pharmacological neutralization of sclerostin, dkk1 or their combination stimulate Wnt signaling and promote IVD structure and (2) determined the extent of the response of the IVD to global, persistent deletion of sost. Nine-week-old C57Bl/6J female mice (n=6-7/grp) were subcutaneously injected 2x/wk for 5.5 wk with scl-Ab (25 mg/kg), dkk1-Ab (25 mg/kg), 3:1 scl-Ab/dkk1-Ab (18.75:6.25 mg/kg) or vehicle (Veh). Separately, IVD of sost KO and WT (wildtype) mice (n=8, grp) were harvested at 16 weeks of age. First, compared to vehicle, scl-Ab, dkk1-Ab and 3:1 scl-Ab/dkk1-Ab similarly increased lumbar IVD height and {beta}-catenin gene expression. Despite these similarities, scl-Ab decreased cellular stress-related heat shock protein gene expressions while neither dkk1-Ab nor scl-Ab/dkk1-Ab altered the same. Genetically and compared to WT, sost KO increased MRI-determined hydration and proteoglycan staining in the IVD. Notably, persistent deletion of sost was compensated by upregulation of dkk1, which consequently reduced the cell nuclear fraction for Wnt signaling transcription factor {beta}-catenin in whole IVD. Lastly, RNA-sequencing pathway analysis confirmed the compensatory suppression of Wnt signaling and determined a reduction of cellular stress pathways. Together, suppression of sost/sclerostin or dkk1 each promote IVD structure by stimulating Wnt signaling, but sclerostin and dkk1 may differentially regulate cellular stress pathways. Ultimately, postmenopausal women prescribed scl-Ab injections to prevent vertebral fracture may also benefit from a restoration of IVD height and health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/449486v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@db2e17org.highwire.dtl.DTLVardef@1af1bbborg.highwire.dtl.DTLVardef@145cc4corg.highwire.dtl.DTLVardef@167a19e_HPS_FORMAT_FIGEXP M_FIG C_FIG Suppression of Wnt signaling inhibitors by genetic or pharmacological approaches promotes intervertebral disc structure and hydration by Wnt signaling. However, persistent activation of Wnt signaling induces a compensatory reduction of Wnt signaling that shifts IVD cells toward a chondrocyte-like (CLC) phenotype. AF: annulus fibrosus, NC: notochordal cell, NP: nucleus pulposus, PG: proteoglycan

bioengineering↗

bioRxiv2020

Sclerostin depletion induces inflammation in the bone marrow of mice

Romosozumab, a humanized monoclonal antibody specific for sclerostin, has been approved for treatment of post-menopausal women with osteoporosis at high risk for fracture. In several Phase III clinical trials, romosozumab decreased the risk of vertebral fractures up to 73% and increased total hip area bone mineral density by 3.2%. Previous work in 12 to 15-week-old sclerostin-knockout (Sost-/-) mice indicated that changes in immune cell development occur in the bone marrow (BM), which could be a possible side effect to follow in human patients. Our overall goal was to define the mechanisms that guide behavior of long-term hematopoietic stem cells (LT-HSCs) after exposure to an irregular BM microenvironment. SOST plays an important role in maintaining bone homeostasis, as demonstrated by the increased ratio of bone volume to total volume observed in Sost-/- mice. Here, we examined the effects of short-term sclerostin depletion in the BM on hematopoiesis in young (8 week-old) mice receiving sclerostin-antibody (Scl-Ab) treatment for 6 weeks, and the effects of long-term Sost-deficiency on wild-type (WT) LT-HSCs transplanted into older (16-22 week-old) cohorts of Sost-/- mice. Our analyses revealed an increased frequency of granulocytes and decreased frequency of lymphocytes in the BM of Scl-Ab treated mice and WT[->]Sost-/- hematopoietic chimeras, indicating myeloid-biased differentiation in Sost-deficient BM microenvironments. This myeloid bias extended to extramedullary hematopoiesis in the spleen and was correlated with an increase in inflammatory cytokines TNF, IL-1 and MCP-1 in the serum of the Sost-/- BM. Additionally, we observed alterations in erythrocyte differentiation in the BM and spleen of Sost-/- mice. Taken together, our current study indicates novel roles for Sost in the regulation of myelopoiesis and control of inflammation in the BM. Our animal studies strongly recommend tracking of hematopoietic function in patients treated with romosozumab.

immunology↗

bioRxiv2019

An Osteocalcin-deficient mouse strain without endocrine abnormalities

Osteocalcin (OCN), the most abundant non-collagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc-) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double knockout (dko) allele (Bglap/2p.Pro25fs17Ter) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their plasma. FTIR imaging of cortical and trabecular bone in these homozygous knockout animals finds alterations in the crystal size and maturity of the bone mineral, hydroxyapatite, compared to wild-type littermates; however, CT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc- allele, blood glucose levels and male fertility in the OCN-deficient mice with Bglap/2pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Potential explanations include effects of each mutated allele on the transcription of neighboring genes, and differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double knockout strain to The Jackson Laboratory for academic distribution.\n\nAuthor SummaryCells that make and maintain bone express proteins that function locally or systemically. The former proteins, such as type 1 collagen, affect the material properties of the skeleton while the latter proteins, such as fibroblast growth factor 23, enable the skeleton to communicate with other organ systems. Mutations that affect the functions of most bone cell expressed proteins cause diseases that have similar features in humans and other mammals, such as mice; for example, brittle bone diseases for type 1 collagen mutations and hypophosphatemic rickets for fibroblast growth factor 23 mutations.\n\nOur study focuses on another bone cell expressed protein, osteocalcin, which has been suggested to function locally to affect bone strength and systemically as hormone. Studies using osteocalcin knockout mice led other investigators to suggest endocrine roles for osteocalcin in regulating blood glucose levels, male fertility, muscle mass, brain development, behavior and cognition. We therefore decided to generate a new strain of osteocalcin knockout mice that could also be used to investigate these non-skeletal effects.\n\nTo our surprise the osteocalcin knockout mice we created do not significantly differ from wild-type mice for the 3 phenotypes we examined: bone strength, blood glucose levels, and male fertility. Our data are consistent with findings from osteocalcin knockout rats, but inconsistent with data from the original osteocalcin knockout mice. Because we do not know why our new strain of osteocalcin knockout mice fails to recapitulate phenotypes previously reported for another knockout mouse stain, we have donated our mice to a public repository so that they can be easily obtained and studied in other academic laboratories.

genetics↗

bioRxiv2019

Osteocytes remodel bone by TGF-β-induced YAP/TAZ signaling

Osteocytes are bone matrix-entombed cells that form an interconnected network of processes called the lacunar/canalicular system, which enables osteocytes to coordinate bone formation and resorption. Osteocytes indirectly regulate osteoblast and osteoclast activity on bone surfaces but also directly resorb and deposit their surrounding bone matrix through perilacunar/canalicular remodeling. However, the molecular mechanisms by which osteocytes control bone remodeling remain unclear. We previously reported that the transcriptional regulators Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-motif (TAZ) promote bone acquisition in osteoblast-lineage cells. Here, we tested the hypothesis that YAP and TAZ regulate osteocyte-mediated bone remodeling by conditional ablation of both YAP and TAZ from mouse osteocytes using 8kb-DMP1-Cre. Osteocyte conditional YAP/TAZ deletion reduced bone mass and dysregulated matrix collagen content and organization, which together impaired bone mechanical properties. YAP/TAZ deletion reduced osteoblast number and activity and increased osteoclast activity. In addition, YAP/TAZ deletion directly impaired osteocyte lacunar/canalicular network remodeling, reducing canalicular density, length, and branching, but did not alter lacunar size or shape. Further, consistent with recent studies identifying TGF-{beta} signaling as a key inducer of perilacunar/canalicular remodeling through expression of matrix-remodeling enzymes, YAP/TAZ deletion in vivo decreased osteocyte expression of matrix proteases Mmp13, Mmp14, and Cathepsin K. In vitro, pharmacologic inhibition of YAP/TAZ transcriptional activity in osteocyte-like cells abrogated TGF-{beta}-induced protease gene expression. Together, these data show that YAP and TAZ act downstream of TGF-{beta} in osteocytes to control bone matrix accrual, organization, and mechanical properties indirectly by coordinating osteoblast/osteoclast activity and directly by regulating perilacunar/canalicular remodeling.

cell biology↗

bioRxiv2017

Skeletal Cell YAP And TAZ Redundantly Promote Bone Development By Regulation Of Collagen I Expression And Organization

The functions of the transcriptional co-activators YAP and TAZ in bone are controversial. Each has been observed to either promote or inhibit osteogenesis in vitro, while their roles in bone development are unknown. Here we report that combinatorial YAP/TAZ deletion from skeletal cells in mice caused osteogenesis imperfecta with severity dependent on targeted cell lineage and allele dosage. Osteocyte-conditional deletion impaired bone accrual and matrix collagen, while allele dosage-dependent deletion from all osteogenic lineage cells caused spontaneous fractures, with neonatal lethality only in dual homozygous knockouts. We identified putative target genes whose mutation in humans causes osteogenesis imperfecta and which contain promoter-proximate binding domains for the YAP/TAZ co-effector, TEAD4. Two candidates, Col1a1 and SerpinH1, exhibited reduced expression upon either YAP/TAZ deletion or YAP/TAZ-TEAD inhibition by verteporfin. Together, these data demonstrate that YAP and TAZ redundantly promote bone matrix development and implicate YAP/TAZ-mediated transcriptional regulation of collagen in osteogenesis imperfecta.

developmental biology↗