Role of Protein Kinase D in Skin Epithelia

描述（由申请人提供）：生理性表皮组织更新的维持由少量干细胞支持，这些干细胞自我更新并在通常认为是单向分化程序中产生分化的后代。然而，我们最近的研究表明，分化的角质形成细胞可以并确实恢复到自我更新的干细胞挑战了这一长期持有的观点。使用标记的表达外皮蛋白的角质形成细胞的谱系追踪研究，我们已经表明分化的角质形成细胞可以被诱导形成多谱系的、带有毛发的皮肤上皮细胞。这些研究表明，分化的角质形成细胞具有可诱导的增殖能力，其响应于来自微环境的线索而被激活。这种可逆性可以在小鼠角质形成细胞的原代培养物中重现，通过将生长停滞的分化的角质形成细胞切换到低钙条件，从而诱导逆转为基底细胞表型和编程。使用该模型，我们确定了蛋白激酶D1（PKD 1）信号通路作为这种去分化的关键。PKD是一种新型的多功能信号传导酶复合物，参与调节许多重要的细胞功能，包括细胞存活、增殖、迁移、分化、膜运输和蛋白质转运。此外，PKD 1还参与多种病理过程，包括应激依赖性心脏肥大、病理性血管生成和肿瘤细胞增殖和转移。令人惊讶的是，尽管所有三种PKD亚型在角质形成细胞中表达，但对这些酶在皮肤中的作用知之甚少。我们的初步数据表明，PKD 1的特异性敲低阻断了分化的角质形成细胞的去分化，而对复制角质形成细胞的培养物中的细胞增殖没有影响。这些数据表明PKD亚型的独特和冗余的作用。我们打算利用本申请的探索性质来研究PKD在皮肤上皮中的作用，并在完整皮肤中建立这种现象的体内相关性。在第一个目标中，我们表征PKD信号在调节皮肤上皮结构和功能中的功能意义。我们将利用PKDloxp/loxp小鼠的可用性来产生条件性PKD 1敲除，以检查PKD 1缺失对皮肤上皮结构和功能的影响。然后，我们使用PKD 1缺陷的角质形成细胞作为一个平台，以确定其他PKD亚型的独特和冗余功能。在第二个目标，我们将扩大使用条件PKD 1基因敲除小鼠，以确定PKD 1的表皮增生/适应性反应的贡献。确定PKD酶在表皮适应性反应中的关键作用将为开发新的治疗策略提供机会，以抑制不受控制的生长，促进损伤后的组织再生，并在衰老期间保持干细胞活性。

DESCRIPTION (provided by applicant): Maintenance of physiological epidermal tissue turnover is supported by a small number of stem cells that self renew and generate differentiated progeny in what is generally thought to be a unidirectional differentiation program. However, our recent studies showing that differentiated keratinocytes can and do revert to self renewing stem cells challenges this long held view. Using lineage tracing studies of labeled involucrin expressing keratinocytes, we have shown that differentiated keratinocytes can be induced to form a multi-lineage, hair-bearing skin epithelia. These studies suggest that differentiated keratinocytes possess an inducible proliferative capacity that is activated in response to cues from the microenvironment. This reversibility could be recapitulated in primary cultures of mouse keratinocytes by switching growth-arrested, differentiated keratinocytes to low calcium conditions thereby inducing reversion to a basal cell phenotype and programming. Using this model we identified the protein kinase D1 (PKD1) signaling pathway as key to this de-differentiation. PKD is a novel, multifunctional, signaling enzyme complex that is involved in the regulation of many important cellular functions including cell survival, proliferation, migration, differentiation, membrane trafficking and protein transport. Moreover, PKD1 is implicated in several pathological processes including stress-dependent cardiac hypertrophy, pathological angiogenesis and tumor cell proliferation and metastasis. Surprisingly, despite expression of all three PKD isoforms in keratinocytes, little is known about the role of these enzymes in skin. Our preliminary data show that specific knockdown of PKD1 blocked dedifferentiation in differentiated keratinocytes while having no effect on cell proliferation in cultures of replicating keratinocytes. These data suggest unique and redundant roles for PKD isoforms. We intend to use the exploratory nature of this application to investigate the role of PKDs in skin epithelia and to establish the in vivo correlate of this phenomenon in intact skin. In the first aim we characterize the functional significance of PKD signaling in regulating skin epithelia structure and function. We will take advantage of the availability of the PKDloxp/loxp mouse to generate a conditional PKD1 knockout to examine the effects of PKD1 loss on skin epithelia structure and function. We then use PKD1-deficient keratinocytes as a platform to identify the unique and redundant functions of other PKD isoforms. In the second aim we will extend the use of conditional PKD1 knockout mice to determine the contribution of PKD1 to the hyperplastic/adaptive responses of epidermis. Defining a critical role for PKD enzymes in epidermal adaptive responses will provide an opportunity to develop novel therapeutic strategies to suppress uncontrolled growth, to promote tissue regeneration after injury and to maintain stem cell activity during aging.