Mechanism Of Receiving And Responding To Hedgehog Morphogenic Gradient

Sonic Hedgehog (Shh) is one of the major developmental morphogens that specify tissue pattern and determine cell fate during embryonic development in vertebrates. Mutations in the Shh pathway and its dysregulation can cause disposition to human congenital anomalies and tumorigenesis. After30years’ intense basic research and clinical testing since the original discovery of Hedgehog in1980, a pathway-specific inhibitor, Erivedge, finally received FDA approval as the first line drug for metastatic basal cell carcinoma in early2012. Thus, further elucidation of Shh signaling mechanism will not only be a core topic of developmental genetics, it will also have practical implications in the clinic.In a developing field, the morphogenic gradient of Shh emanated from the organizing cells provides positional cues along a specific axis to cells in the tissue primordium, which must have the ability to discern subtle changes in the gradient and generate transcriptional responses accordingly. However, Shh signaling entails a highly complex mechanism, with many key regulatory nodes still being poorly understood. On the cell surface, Shh signaling is mediated by two transmembrane receptors, Patched (Ptch) and Smoothened (Smo). Ptch is the Shh receptor and has the ability to physically interact with Shh, but it is an inhibitory regulator of Shh signaling. In contrast, Smo does not bind Shh, and is an active regulator of Shh signaling. In regions away from the source of Shh signal, Ptch suppresses the intrinsic activity of Smo through yet unknown mechanisms, and promotes Smo turnover via the endocytic pathway. In regions juxtaposition to the source of Shh signal, Shh binds Ptch and thereby alleviates its inhibition of Smo to activate downstream signaling events. Downstream from Smo, expression of Shh target genes is regulated by transcription factors Gill, Gli2and Gli3. In the presence of no or low level of Shh, Gli2and Gli3undergo a ubiquitin-proteasome pathway-mediated proteolytic processing that converts them into truncated transcription repressors (Gli2R and Gli3R), which traverse into the nucleus to actively inhibit Shh target gene expression. In the presence of high level of Shh, Gli processing is blocked, allowing Gli2and Gli3to be stablized as full length transcription activators (Gli2A and Gli3A) for target gene expression. Glil is an auxiliary activator that does not undergo the proteolytic processing event. Thus, a gradient of transcriptional activities formed by a combinational mixing of full length Glil, Gli2A and Gli3A activators along with truncated Gli2R and Gli3R repressors underpins the proportional responses by recipient cells to the variant positional cues carried in the Shh morphogenic gradient.In this dissertation, I focused my study on the regulation of a Gli-binding protein, Suppressor of Fused (Sufu), which is an essential downstream inhibitor of Shh signaling. Sufu inhibits the expression of Shh target genes through physical binding to Gli proteins. Our results showed that Shh promotes Sufu degradation through the ubiquitin-proteasome system, thereby alleviating its repression of target gene expression. I found that Sufu was degraded rapidly in certain cancer cell lines with aberrant Shh activation, while treating these cells with Smo antagonists decreased the rate of Sufu degradation. I also showed that Sufu undergoes ubiquitin-proteasomal degradation in response to Shh signaling in freshly isolated mouse embryonic fibroblasts and embryonic tissues, suggesting that Shh also controls Sufu stability under physiological conditions in vivo. We identified an ubiquitin attachment site at K257on Sufu by liquid chromatography and tandem mass spectrometry analysis, and showed that the K257R mutant of Sufu is more stable and has a more potent repression activity towards Glil-mediated transcriptional response and proliferation in NCI-H322M lung cancer cells than its wild type counterpart.A marked characteristics of vertebrate Shh signaling is its absolute dependence on the primary cilium, a microtubule-based cell protrusion present in every interphase cell. Since the original implication of primary cilia in Shh signaling by Kathryn Anderson in2003, multiple components of the Shh pathway, including Sufu, have been localized in primary cilia. In cooperation with other members of the lab, I found that Shh induces ciliary localization of endogenous Sufu in cultured fibroblasts, and the transport of Sufu into or out of cilia likely occurs in the form of a Sufu-Gli2/Gli3complex and depends on Smo activity. Moreover, the ciliary transport of Sufu is closely associated with its Shh-induced degradation. I further showed that Sufu is phosphorylated at Ser-346and Ser-342by PKA and GSK3β sequentially, and phosphorylation at this dual site stabilizes Sufu against Shh-induced degradation, and prolongs the stay of Sufu in the cilia, thus implying a functional significance of Sufu trafficking into the primary cilium.To further investigate the Shh signaling mechanisms, we conducted a targeted siRNA-based screen for the role of ubiquitin modification among the HECT-domain E3ligases, and identified Smurfl and Smurf2as potential regulators of Shh signaling. My results indicated that simultaneous knockdown of Smurfl and Smurf2or homologous inactivation of both alleles in primary mouse embryonic fibroblasts using cre-expressing adenoviruses blocks the Shh-induced ciliary localization of Smo. The HECT-domain E3ligases recognize a "PY" motif in the substrates and catalyze their ubiquitin modification by binding to such sites. By analyzing protein sequences of known Shh pathway components, I found that mouse and human Ptchl both have two PY motifs in their intracellular domains, respectively. Mutating either one of these two "PY" motifs or both lead to a conspicuous accumulation of the mutant Ptchl in the primary cilia, compared to the wild type counterpart. Further experiments indicated that either Smurfl or Smurf2can bind Ptchl, and the "PY" motif likely constitutes a cis-acting signal in Ptchl for export out of the primary cilium, followed by endocytosis and lysosome-mediated degradation. To address the physiological relevance of Smurfl and Smurf2regulation of Ptch, I isolated the primary granular neuron precursor cells (GNPCs) from the cerebella of P6mouse pups and cultured them in vitro. Knockdown of both Smurfl and Smurf2markedly curtailed the proliferation of GNPCs sustained by Shh.In my thesis study, I investigated how Shh signaling regulates post-translational modification and intracellular transport of Ptch and Sufu for alleviating their respective repression of the pathway activity, and partially elucidated the mechanisms by which the receiving cells perceive and respond to the morphognic gradient of Shh. The key sites and protein molecules identified in the post-translational modifications of Sufu and Ptchl should generate new insight and can be used as novel targets in search for therapeutic compounds targeting the Shh signaling pathway.