Physiological and pathological functions of E3 ubiquitin ligases Smurfs

Smad泛素调节因子（Smurfs）是E3泛素连接酶的HECT家族的成员。两个Smurf，Smurf1和Smurf2，基于它们通过促进Smads上的泛素修饰来调节TGF-β/BMP信号传导的活性而被鉴定;然而，随后的研究已经扩展了Smurf底物的库，以包括TGF-β/BMP通路之外的蛋白质，如RhoA，Runx2和MEKK 2。为了解决蓝精灵的生理功能，我们已经产生了缺乏Smurf1或Smurf2的小鼠。我们发现，这两个旁系同源的E3泛素连接酶在胚胎发生和维持成人生理稳态的共同和独特的功能。我们早期对Smurf 1基因敲除小鼠的研究揭示了调节成骨细胞功能和骨稳态的新机制，这表明靶向Smurf 1可能被证明是治疗骨质疏松症中与年龄相关的骨丢失的有效策略。我们随后对Smurf2基因敲除小鼠的研究澄清了文献中关于Smurf调节TGF-β信号传导的矛盾报道，表明Smurf2确实具有抑制作用，但它是通过单泛素化减弱Smad3活性而不是促进其降解来实现的。由于两个Smurf的缺失导致胚胎死亡，我们创建了条件性Smurf2敲除等位基因，这使得我们能够通过使用cre重组酶的组织特异性表达完全去除所需组织中的所有Smurf活性来研究Smurf的生理功能，并从晚期胚胎或成人组织获得条件性Smurf无效细胞。使用Smurf双敲除细胞，我们确定Smurfs作为E3连接酶，其通过泛素修饰控制Sonic hedgehog（Shh）受体Patched1的内吞周转。我们证明，这种调节是至关重要的激活Shh信号在细胞培养试验和维持小脑颗粒神经元前体的Shh依赖性增殖。我们对Smurf2基因敲除小鼠的研究还意外地发现了Smurf2作为肿瘤抑制因子的一种以前未被认识的功能，这种功能通常通过控制组蛋白修饰的表观遗传景观来维持基因组的稳定性。我们发现Smurf2与一组调节DNA复制、染色质重塑和组蛋白修饰的蛋白质相互作用。我们目前正在验证这些发现，在体外和体内的方法，并调查这些相互作用在基因组稳定性和肿瘤发生的生物学相关性。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，但在细胞质中表达增加。Smurf1表达在乳腺癌细胞的细胞质中也上调。Smurf1和Smurf2的过表达促进裸鼠模型中的转移，并诱导乳腺癌细胞的上皮-间充质转化、迁移和侵袭，表明Smurfs在乳腺癌进展中起重要作用。我们目前正在研究Smurf1和Smurf2促进癌细胞迁移和侵袭的机制。

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.