HIF1A function and regulation
A,B. Detection of fluorescence in frozen sections of NP isolated from E15.5 (A) and 1 month (B) HIF-1αf/f (a,e,i), HIF-1αf/f;mTmG (b,f,j), Foxa2Cre;HIF-1αf/+;mTmG (c,g,k) and Foxa2Cre;HIF-1αf/f;mTmG (d,h,l) mice, respectively. Red fluorescence (a-d), green fluorescence (e-h) and merged filters (i-l) are shown. Bar = 100 µm. Cells with deletion of HIF1A (green) disappear post natally
Since establishing an independent research program at Jefferson, one of the major focuses of my laboratory has been to investigate the importance of local environmental factors in regulating cell function in the intervertebral discs--the soft tissue between vertebrae that serves as shock absorbers in the spine. Back pain is a ubiquitous condition and the second most prevalent neurological ailment in the United States, costing the country upwards of $100 billion each year. This painful syndrome is closely linked to degenerative changes that impair the function of the intervertebral disc. My lab’s work was the first to show a differential expression of HIF-1 in nucleus pulposus (NP) tissue and not in the annulus fibrosus and cartilage endplate within the intervertebral disc, as was commonly believed. Subsequent work clearly showed that NP cells exhibit uniquely stabilized HIF-1 and that this stability is controlled through the coordinated actions of prolyl-4-hydraxylases (PHD)1-3. Interestingly, HIF-α proteins show a novel mode of regulation: PHD2, but not PHD3, controls HIF-1α turnover to an extent while HIF-2α resists PHD-mediated degradation. We have also conclusively demonstrated that HIF-1 function in disc cells is refractory to regulation by FIH-1, an asparaginyl hydroxylase known to control HIF transcriptional activity. Our works have defined several other regulators that control HIF-1 activity, including HSP70 and matricellular protein CCN2, and demonstrated a unique HIF-1 transcriptional program in this hypoxic tissue. We recently generated NP-specific HIF-1α null mice and demonstrated the vital role HIF-1 plays in cell survival and function. Overall, our work has firmly established that HIF-1 is indispensable in (and a master regulator of) NP cell adaptation to their unique hypoxic niche.
Inflammation in the intervertebral disc.
Safranin O/Fast Green staining of 8-week-old mouse caudal intervertebral disc.
We are exploring the mechanisms by which inflammatory cytokines promote disc degeneration and inflammation. We found an unexpected synergism between two seemingly unrelated protein families: heparan sulfate proteoglycans, syndecan and the key catabolic molecules. We discovered that inflammatory cytokines upregulate syndecan-4 expression and influence the expression and activity of ADAMTS as well as MMP-3, both of which promote the breakdown of proteoglycan and the collagen-rich matrix of the disc. While these events probably represent an initiating factor leading to degeneration, we also noted that the inflammatory cytokines promote chemokine expression by disc cells and are thus instrumental in chemotaxis and macrophage infiltration. This finding led to the generation of a more comprehensive theory encompassing feed-forward events triggering back pain as well as the amplification of inflammatory events that promote disc degeneration. We also discovered novel molecular mechanisms that control the activity of cytokines and of NF-κB signaling pathway, a central mediator of inflammatory signaling. Our work shows the existence of a prominent cross talk between hypoxia and inflammatory pathways in the disc. The members of PHD2 and 3 that control HIF-α degradation promote the transcriptional activity of a prominent NF-κB family member p65/RelA. This finding has both high clinical and translational significance as several lead molecules targeting PHD function are in clinical trials and may represent a novel way of controlling disc inflammation.
TonEBP Function and Regulation
Another major focus of the laboratory is to understand how the hyperosmotic niche of the nucleus pulposus, regulates functions of resident cells. We made a significant advancement with an observation that the osmotic balance in NP cells is regulated by the activities of the transcription-factor tonicity-regulated Enhancer Binding Protein (TonEBP or NFAT5). Our studies showed that this protein is important in regulating survival and apoptosis in NP cells under hypertonic and mechanically stressed conditions. We showed that upstream MAPK signaling (in particular ERK), in an isoform-specific fashion, changes Ca2+ levels and that TGFβ family members control activity of TonEBP in disc cells. Moreover, we have shown that TonEBP controls expression of a number of critical molecules involved in extracellular matrix homeostasis, including aggrecan and GlcAT-I, an enzyme required for GAG synthesis. We have also shown that TonEBP controls water homeostasis in the disc by controlling AQP2 water channel levels. More recently, we have started to investigate role of TonEBP in controlling broader transcriptional programs in NP cells, using RNA-Sequencing technology. We have identified many novel pathways, including inflammation related genes, as TonEBP targets in NP. This work has important clinical implications because disc degeneration is characterized by changes in proteoglycan status and a loss of bound water and tissue-osmotic pressure, which decrease the disc’s ability to absorb biomechanical forces. This research may lead to new restorative therapies targeting TonEBP function through the use of small molecules, a current area of research in my laboratory.
Regulation of metabolic systems in NP cells
NP cells retrovirally transduced with tandem mCherry-EGFP-LC3B construct, showing yellow/green autophagosomes and red autolysosomes.
Another major goal in our lab is to understand how the disruption of critical metabolic systems in NP cells can contribute to the pathogenesis of age-related disc degeneration. Given that NP cells reside in a physiologically hypoxic niche and rely primarily on glycolysis for ATP generation, these cells reside in an environment characterized by acidic pH. Pathologically acidic pH in NP cells has been shown to lead to matrix catabolism and decreased glucose uptake; therefore NP cells rely on robust pH-regulating systems to maintain the optimal pH for cell survival. HIF-1α, in particular, regulates the expression of many genes critical to the survival of NP cells. We hypothesize that the HIF-1α- Carbonic anhydrase axis is critical for pHi regulation in NP cells. In addition, we are interested in determining the function and regulation of autophagy in NP cells in their hypoxic niche, and the implication of this cellular process in the pathogenesis of disc degeneration. We have shown that, unlike many other cell types, NP cells under hypoxia modulate autophagy independently of HIF-1α. Surprisingly, NP cells carry out a highly unique, non-canonical autophagy through MTOR independent pathway, regardless of the oxygen tension. This suggests that autophagy may have a non-metabolic role in NP cells.