Role of Protein Kinase D in Skin Epithelia

蛋白激酶 D 在皮肤上皮细胞中的作用

• 批准号: 8331613
• 负责人: SOOSAN GHAZIZADEH
• 金额: $ 18万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8331613
• 关键词: Adherence; Aging; Basal Cell; Beds; Biological Process; Blocking Antibodies; Calcium; Cardiac; Cell Proliferation; Cell Survival; Cell physiology; Cells; Chemicals; Cues; Cutaneous; Cyclin D1; DNA biosynthesis; Data; Development; Differentiation and Growth; Down-Regulation; E-Cadherin; Enzymes; Epidermis; Epithelium; Exploratory/Developmental Grant; Future; Gene Transfer; Genetic; Goals; Growth; Growth Factor; Hair; Heart Hypertrophy; Hela Cells; Hyperplasia; Implant; Injury; Knock-out; Knockout Mice; Label; Maintenance; Mammals; Membrane Protein Traffic; Modeling; Monitor; Multienzyme Complexes; Mus; Nature; Neoplasm Metastasis; Normal Range; Nude Mice; Pathologic Neovascularization; Pathologic Processes; Phenotype; Phorbol Esters; Physiological; Play; Process; Proliferating; Protein Isoforms; Protein Kinase; Regulation; Repression; Role; Signal Pathway; Signal Transduction; Skin; Skin Carcinogenesis; Stem cells; Stimulus; Stress; Structure; Testing; Thick; Tissues; Tumor Promoters; Vertebrates; Wound Healing; angiogenesis; base; in vivo; insight; involucrin; keratinocyte; migration; mutant; neoplastic cell; novel; novel strategies; novel therapeutics; premature; programs; protein kinase D; protein transport; research study; response; self-renewal; skin regeneration; stem cell population; tissue regeneration; tumor; wound

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.

描述（由申请人提供）：生理表皮组织更新的维持是由少量干细胞支持的，这些干细胞在通常被认为是单向分化程序中自我更新并产生分化的后代。然而，我们最近的研究表明，分化的角质形成细胞可以并且确实恢复为自我更新的干细胞，这对这一长期持有的观点提出了挑战。通过对标记的表达外皮蛋白的角质形成细胞进行谱系追踪研究，我们发现分化的角质形成细胞可以被诱导形成多谱系、有毛发的皮肤上皮。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，该能力可响应微环境的提示而被激活。通过将生长停滞、分化的角质形成细胞切换到低钙条件，从而诱导向基底细胞表型和编程的回复，可以在小鼠角质形成细胞的原代培养物中重现这种可逆性。使用该模型，我们确定了蛋白激酶 D1 (PKD1) 信号通路是这种去分化的关键。 PKD 是一种新型多功能信号酶复合物，参与许多重要细胞功能的调节，包括细胞存活、增殖、迁移、分化、膜运输和蛋白质运输。此外，PKD1 还涉及多种病理过程，包括应激依赖性心脏肥大、病理性血管生成以及肿瘤细胞增殖和转移。令人惊讶的是，尽管所有三种 PKD 同工型都在角质形成细胞中表达，但人们对这些酶在皮肤中的作用知之甚少。我们的初步数据表明，PKD1 的特异性敲低可阻止分化角质形成细胞的去分化，同时对复制角质形成细胞培养物中的细胞增殖没有影响。这些数据表明 PKD 同工型具有独特且冗余的作用。我们打算利用该应用的探索性来研究 PKD 在皮肤上皮细胞中的作用，并建立完整皮肤中这种现象的体内相关性。第一个目标是描述 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 PKD1 敲除，以检查 PKD1 缺失对皮肤上皮结构和功能的影响。然后，我们使用 PKD1 缺陷的角质形成细胞作为平台来识别其他 PKD 同种型的独特和冗余功能。在第二个目标中，我们将扩大条件性 PKD1 敲除小鼠的使用，以确定 PKD1 对表皮增生/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以及在衰老过程中维持干细胞活性。