Role of Protein Kinase D in Skin Epithelia

描述(申请人提供)：维持生理性表皮组织的周转是由少量干细胞支持的，这些干细胞自我更新并产生分化的后代，这通常被认为是单向分化计划。然而，我们最近的研究表明，分化的角质形成细胞可以而且确实可以恢复到自我更新的干细胞，这对这一长期持有的观点提出了挑战。通过对标记的表达总蛋白的角质形成细胞的谱系追踪研究，我们发现分化的角质形成细胞可以被诱导形成多谱系的、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，这种能力是在微环境的提示下被激活的。这种可逆性可以在原代培养的小鼠角质形成细胞中重现，方法是将生长受阻的分化角质形成细胞切换到低钙条件，从而诱导回复到基本细胞表型和编程。利用这个模型，我们发现蛋白激酶D1(PKD1)信号通路是这种去分化的关键。PKD是一种新型的多功能信号转导酶复合体，参与调节多种重要的细胞功能，包括细胞存活、增殖、迁移、分化、膜转运和蛋白质转运。此外，PKD1参与了应激性心肌肥大、病理性血管生成、肿瘤细胞增殖和转移等多种病理过程。令人惊讶的是，尽管在角质形成细胞中表达了所有三种PKD亚型，但对这些酶在皮肤中的作用知之甚少。我们的初步数据显示，在复制的角质形成细胞的培养中，PKD1的特异性敲除阻止了分化角质形成细胞的去分化，而对细胞的增殖没有影响。这些数据表明了PKD亚型的独特和多余的作用。我们打算利用这一应用的探索性性质来研究PKD在皮肤上皮细胞中的作用，并建立在完整皮肤中这一现象的体内相关性。在第一个目的中，我们表征了PKD信号在调节皮肤上皮细胞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有条件的PKD1基因敲除，以检测PKD1缺失对皮肤上皮细胞结构和功能的影响。然后，我们使用PKD1缺失的角质形成细胞作为平台，以确定其他PKD亚型的独特和冗余功能。在第二个目的中，我们将扩展使用有条件的PKD1基因敲除小鼠来确定PKD1对表皮的增殖/适应性反应的贡献。明确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的活性。

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.