While the hematopoeitic stem cell is a clearly defined cell type, efforts to identify a universal and standard mesenchymal stem cell have not been successful. Instead, mesenchymal progenitors are not equivalent between tissues, and harbor numerous differences in the pathways regulating their lineage up to the mesenchymal progenitor stage. Our laboratory investigates the identities of mesangial progenitors resident in bone, kidney, and adipose. To do this we use the mouse as a genetic model system and interrogate both extrinsic signaling pathways that modulate their differentiation as well as key intrinsic transcriptional processes.
Specialized Terms: mesenchymal cell differentiation; kidney development; renal osteodystrophy; transcription; osteoprogenitors; adipocyte; osteoblast; pericyte; mesangial cell; skeletal homeostasis; MSC; perivascular cells
My research focuses on the role of the transcription factor (TF) Early B cell factor 1 (EBF1) in cellular fate determination and disease. EBF1 is one of four orthologous proteins that exist in vertebrates. This family of TFs is unique owing to the unusual structure of their DNA-binding domains. Working as homo-or hetero-dimers the EBF proteins contact DNA though an atypical zinc finger, and secondly through an IPT/TIG-like domain. Resultantly, they have an extremely flexible response element that makes in silico prediction of potential binding sites unreliable. Although EBF1 was named for its essential role in B cell lymphopoiesis, it has since been found to be essential to the formation and function of white adipose, neuronal subsets, vascular support populations, and as I discovered, kidney development. My research is focused on areas outside of the immune system where EBF1 is known to function; mesenchymal cell fate into the adipocyte lineage within the bone marrow contrasted against the peripheral adipose, and development and maintenance of the kidney glomerulus. As more is revealed about the actions of EBF1 in these disparate locations we are discovering that some conserved actions of EBF1 appear to be implicated in both processes.
EBF1 in Bone and Adipogenesis
I began working with EBF1 because this TF has an important role in the development of peripheral white adipose, where it promotes the mesenchymal transition to the adipocyte cell fate. We had originally hypothesized that EBF1 would participate in the formation of marrow adipose similarly to how it functions in peripheral white adipose (increasing bone density as progenitors were shunted away from the adipogenic fate to an osteoblastic one). Instead we were the first to report that EBF1-dependence proved to be a unique bifurcation point in cellular regulation that is not shared by these two adipocyte cell types. While EBF1 knockout mice are lipodystrophic with pronounced decreases in the perigonadal and mesenteric white depots, which are more heavily dependent upon EBF1 expression than subcutaneous depots, marrow fat formation is not inhibited and in fact increases. There is a very complex phenotype present in the global knockout mice that can cause non-cell intrinsic changes in marrow composition. Targeted deletion of EBF1 from the limb bud and all the mesenchymal lineage cells of the long bones results in bones that have marked changes in the bone vasculature and a pronounced increase in the number of marrow adipocytes present. We have traced this back to the pericyte subsets of the stroma and are currently identifying the transcriptional targets of EBF1 that make these marrow progenitors respond so differently than their peripheral white counterparts. At the same time there is an accumulation of marrow adipocytes that arise from these pericytes and a decrease in the commitment of these progenitors into the osteoblast lineage. Preliminary data suggest that the shift in lineage allocation may be a conserved consequence of EBF1 in these mesenchymal progenitors of both white adipose and in bone, and relate back to the direct function of EBF1 as a transcriptional regulator of SREBF1. I am submitting an R01 for this October deadline to the NIH to investigate these cells and the physiological consequences to the loss of EBF1.
In the peripheral white adipose I have discovered that in the absence of EBF1 white adipose is also resistant to the formation of metabolically favorable beige adipocytes. Interestingly, several stimuli that increase beigeing of the white fat also promote formation of marrow adipocytes, yet these two populations respond very differently to the absence of EBF1. In addition to an absence of entry into the beige cell fate my work discovered that peripheral white adipocytes lacking EBF1-expression are hypoproliferative, but also hypertrophic. EBF1 expression varies in peripheral adipose tissue in the human population. The consequence of this variation in expression is that those affected individuals with the lowest expression of EBF1 do not show significant change in body mass index, but do have hypertrophic adipocytes, display abrogated lipolysis, and are more insulin resistant than the individuals with normal EBF1 expression. We were also able to demonstrate that a conserved constellation of metabolic phenotypes (increased basal lipolysis, decreased stimulated lipolytic response, adipocyte hypertrophy and insulin resistance) occurs in global Ebf1 heterozygous mice. Mechanistic investigations that we have undertaken and which are still ongoing implicate EBF1 in the regulation of some key adipogenic receptors and proteins including the TF SREBP1 and beta-adrenergic receptors.
EBF1 in kidney glomerular function
My discovery of a role for EBF1 in the kidney arose from our investigations into its function in the bone. Paradoxically, the EBF1 global knockout mice had low bone density, but also exhibited high circulating levels of the anabolic marker Osteocalcin. Osteocalcin is normally an accurate marker of bone generation, until the integrity of the glomerular filtration barrier of the kidney is compromised. In the absence of EBF1 the mice have a congenital malformation of the glomerulus that results in renal failure. EBF1-deficient mice have thinned cortices, with poorly developed peripheral glomeruli and islands of undifferentiated mesenchyme that persist throughout adulthood. This was a discovery of a wholly novel role for EBF1 in physiology and development. Using targeted deletion our work has revealed that it is the mesangial cells and the stromal lineage (a mesenchymal derived cell population) that control capillary tuft branching. My research into EBF1 as an essential TF for proper maturation of the outer cortex and the glomerular tuft has revealed that EBF1 functions at the very last stages of capillary tuft development, a point of differentiation where the transcriptional and cytochemical processes involved are not well understood. It is interesting to note that both in the bone and in the kidney, although the exact consequence of the loss of EBF1 varies, both roles for EBF1 involve support and proliferation of an associated vascular network. Through investigation of the biological actions regulating EBF1 expression, and identification of the pathways affected by the actions of EBF1, I hope to better understand renal development and kidney disease with the goal of improving treatment strategies in the patient population afflicted with these debilitating and progressive conditions. This would be a valuable inroad for therapies because slowing and or reversal of glomerulosclerosis requires a resumption of capillary branching along with the resorption of fibrotic matrix. Investigation into the role of EBF1 in the regulation of the formation of this unique vascular bed will provide needed and still unknown insight into the processes that govern its formation.
Beyond this first action of EBF1 in the mesangial cell, we have also identified that there is a second role for this TF in the glomerular podocyte. Preliminary data from my laboratory has demonstrated that mice that lack EBF1 expression in only the podocytes are protected from kidney damage. While damage to all three types of glomerular cell can disrupt the filtration barrier, it is the limited potential of the podocyte to proliferate that is thought to precipitate sclerosis. EBF1 participates in the normal damage response of podocytes, and in its absence the podocytes are protected from effacement, sloughing, and consequently sclerosis. While we now understand that damage to and loss of podocytes from the capillary tuft is the event that precipitates glomerulosclerosis, the mechanisms controlling these processes are largely unknown. My pilot grant from the Yale O’Brien Center focused on identifying the relative protective contribution of EBF1 expression to the progression of glomerulosclerosis in both a rapidly progressing and slower acting but reversible model of glomerular injury, with the aim of identifying the signaling pathways and underlying biology employed by the podocyte to regulate both sclerotic progression and regression.
Connections between bone and kidney
There appears to be a conserved function of EBF1 between the bone and kidney where it is needed within mesenchymal vascular support cells in both locations. Interestingly, as we obtain more and more data from our injury models and the actions of EBF1 in adipogenic differentiation conserved metabolic pathways and key processes are consistently arising as transcriptional targets and downstream consequences of the loss of EBF1 in these non-immune cells. In fact, even our podocyte data has overlapping transcriptional targets and pathways that appear to be important for EBF1 regulation of beigeing as well as maintenance of podocyte integrity after kidney injury. These are molecular pathways that were not known at all to participate in the cellular processes that we are using EBF1 to investigate, and are opening new avenues to advance our understanding of these developmental processes and disease states regulated by EBF1.
Bone and Bones; Bone Diseases; Bone Diseases, Metabolic; Fractures, Ununited; Glomerular Filtration Rate; Glomerular Mesangium; Glomerulosclerosis, Focal Segmental; Juxtaglomerular Apparatus; Orthopedics; Osteoblasts; Chronic Kidney Disease-Mineral and Bone Disorder; Transcriptional Activation; Adipocytes; Pericytes; Podocytes; Mesenchymal Stem Cells
Aging; Chronic Diseases; Metabolism