Physiological and pathological functions of E3 ubiquitin ligases Smurfs

Smad泛素调节因子(SNURF)是E3泛素连接酶Hect家族的成员。根据它们通过促进Smads上的泛素修饰来调节转化生长因子-β/骨形态发生蛋白信号的活性，确定了两个蓝精灵，即SMurf1和SMurf2；然而，随后的研究扩大了SMurf底物的研究范围，将RhoA、Runx2和MEKK2等蛋白质包括在转化生长因子-β/骨形态发生蛋白途径之外。为了解决蓝精灵的生理功能，我们培育了缺乏蓝精灵1或蓝精灵2的小鼠。我们发现这两个相似的E3泛素连接酶在胚胎发育和维持成人生理平衡方面既有共同的功能，也有独特的功能。我们早期对S-1基因敲除小鼠的研究揭示了一种调节成骨细胞功能和骨稳态的新机制，提示靶向S-1可能被证明是治疗骨质疏松中年龄相关性骨丢失的有效策略。我们随后对SMurf2基因敲除小鼠的研究澄清了文献中关于SMurf调控转化生长因子-β信号的相互矛盾的报告，表明SMurf2确实具有抑制作用，但它是通过单一泛素化来减弱Smad3的活性，而不是像之前报道的那样促进其降解。由于这两个蓝精灵的缺失会导致胚胎死亡，我们创造了一个条件性SMurf2基因敲除等位基因，它使我们能够通过组织特异性表达cre重组酶完全去除所需组织中的所有蓝精灵活性来研究蓝精灵的生理功能，并从晚期胚胎或成人组织中获得条件性SMurf零细胞。利用SMurf双基因敲除细胞，我们鉴定SMurf是E3连接酶，通过泛素修饰控制Sonic hedgehog(Shh)受体Patched1的内吞翻转。我们证明，在细胞培养实验中，这种调节对于Shh信号的激活以及维持依赖Shh的小脑颗粒神经元前体的增殖都是至关重要的。我们对SMurf2基因敲除小鼠的研究还导致了一个意想不到的发现，即SMurf2作为一种肿瘤抑制因子，通常通过控制组蛋白修饰的表观遗传格局来维持基因组稳定，这一功能以前并不为人所知。我们发现SMurf2与一组蛋白质相互作用，这些蛋白质调控DNA复制、染色质重塑和组蛋白修饰。我们目前正在使用体外和体内方法验证这些发现，并调查这些相互作用在基因组稳定性和肿瘤发生中的生物学相关性。与SMurf2的肿瘤抑制功能一致，我们发现SMurf2在乳腺癌细胞的细胞核中表达减少，但在细胞质中的表达增加。S-1的表达也在乳腺癌细胞的细胞质中上调。SMurf1和SMurf2的过表达促进了裸鼠模型的转移，并诱导了乳腺癌细胞向上皮间充质的转化、迁移和侵袭，提示SMurfs在乳腺癌的发展中发挥着重要作用。我们目前正在研究S-1和S-2促进癌细胞迁移和侵袭的作用机制。

Smad ubiquitin regulatory factors (Smurfs) are members of the HECT family of E3 ubiquitin ligases. Two Smurfs, Smurf1 and Smurf2, were identified based on their activities to modulate TGF-beta/BMP signaling by promoting ubiquitin modification on Smads; however, subsequent studies have expanded the repertoire of Smurf substrates to include proteins such as RhoA, Runx2 and MEKK2 outside the TGF-beta/BMP pathway. To address the physiological function of Smurfs, we have generated mice lacking either Smurf1 or Smurf2. We found that these two paralogous E3 ubiquitin ligases have both common and unique functions during embryogenesis and in maintaining adult physiological homeostasis. Our earlier work on characterizing Smurf1 knockout mice revealed a novel mechanism in regulating osteoblast function and bone homeostasis, suggesting that targeting Smurf1 may prove to be an effective strategy for treating age-related bone losses in osteoporosis. Our subsequent work on characterizing Smurf2 knockout mice clarified contradictory reports in the literature about Smurf regulation of TGF-beta signaling by showing that Smurf2 indeed has an inhibitory role, but it does so by attenuating Smad3 activity through mono-ubiquitination rather than promoting its degradation as previously reported. Because loss of both Smurfs leads to embryonic lethality, we have created a conditional Smurf2 knockout allele, which enabled us to investigate physiological functions of Smurfs by completely removal of all Smurf activities in desired tissues using tissue-specific expression of cre recombinase and obtain conditional Smurf null cells from late stages embryos or adult tissues. Using Smurf double knockout cells, we identified Smurfs as the E3 ligases that control endocytic turnover of Sonic hedgehog (Shh) receptor Patched1 via ubiquitin modification. We demonstrated that this regulation is crucial for the activation of Shh signaling both in cell culture assays and in sustaining the Shh-dependent proliferation of cerebellar granule neuron precursors. Our study of Smurf2 knockout mice also led to an unexpected discovery of a previously unrecognized function of Smurf2 as a tumor suppressor that normally maintains genomic stability by controlling epigenetic landscape of histone modifications. We found that Smurf2 interacts with a group of proteins that regulate DNA replication, chromatin remodeling and histone modification. We are currently validating these findings using in vitro and in vivo approaches and to investigate the biological relevance of these interactions in genomic stability and tumorigenesis. Consistent with the tumor suppresser function of Smurf2, we found that Smurf2 expression is decreased in the nucleus but increased in the cytoplasm of breast cancer cells. Smurf1 expression is also upregulated in the cytoplasm of the breast cancer cells. Overexpression of Smurf1 and Smurf2 promotes metastasis in nude mouse models and induces epithelial-to-mesenmchymal transition, migration, and invasion of breast cancer cells, suggesting that Smurfs play important roles in breast cancer progression. We are currently studying the mechanism underpinning the role of Smurf1 and Smurf2 promoting cancer cell migration and invasion.