ORIGINAL ARTICLE Hair follicle defects and squamous cell carcinoma formation in Smad4 conditional knockout mouse skin W Qiao 1 , AG Li 2 , P Owens 2 , X Xu 1 , X-J Wang 2 and C-X Deng 1 1 Genetics of Development and Disease Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA and 2 Department of Otolaryngology, Oregon Health and Science University, Portland, OR, USA Smad4 is the common mediator for TGFb signals, which play important functions in many biological processes. To study the role of Smad4 in skin development and epidermal tumorigenesis, we disrupted this gene in skin using the Cre-loxP approach. We showed that absence of Smad4 blocked hair follicle differentiation and cycling, leading to a progressive hair loss of mutant (MT) mice. MT hair follicles exhibited diminished expression of Lef1, and increased proliferative cells in the outer root sheath. Additionally, the skin of MT mice exhibited increased proliferation of basal keratinocytes and epidermal hyper- plasia. Furthermore, we provide evidence that the absence of Smad4 resulted in a block of both TGFb and bone morphogenetic protein (BMP) signaling pathways, includ- ing p21, a well-known cyclin-dependent kinase inhibitor. Consequently, all MT mice developed spontaneous malignant skin tumors from 3 months to 13 months of age. The majority of tumors are malignant squamous cell carcinomas. A most notable finding is that tumorigenesis is accompanied by inactivation of phosphatase and tensin homolog deleted on chromosome 10 (Pten), activation of AKT, fast proliferation and nuclear accumulation of cyclin D1. These observations revealed the essential functions of Smad4-mediated signals in repressing skin tumor formation through the TGFb/BMP pathway, which interacts with the Pten signaling pathway. Oncogene (2006) 25, 207–217. doi:10.1038/sj.onc.1209029; published online 19 September 2005 Keywords: squamous cell carcinoma; hair follicle cycle; Pten; AKT; cyclin D1 Introduction The TGFb superfamily consists of more than 40 structurally related proteins that can be divided into several subfamilies, including TGFb, bone morpho- genetic proteins (BMPs) and activins/inhibins (Massa- gue, 1998; Derynck et al., 2001; Wakefield and Roberts, 2002). TGFb signaling plays many important functions in numerous biological processes. Some of the areas that are subjected to extensive investigation include skin development and neoplasia. Many members of TGFb superfamily and their receptors are expressed in skin and developing hair follicles (Li et al., 2003a). It has been shown that alterations in BMP signaling pathway resulted in abnormal hair follicle growth (Kobielak et al., 2003; Li et al., 2003a; Andl et al., 2004; Ming Kwan et al., 2004; Yuhki et al., 2004). In addition, alterations in TGFb signaling result in skin tumorigen- esis (Wang, 2001). For instance, targeted disruption of TGFb1 resulted in tumorigenesis of v-rasHa oncogene- transfected keratinocytes after they were grafted into nude mice (Glick et al., 1994). Consistently, transgenic mice expressing a dominant-negative type II TGFb receptor exhibited epidermal hyperproliferation (Wang et al., 1997) and were more sensitive to chemically induced carcinogenesis (Amendt et al., 1998; Go et al., 1999). Benign hair follicle tumors were also observed in mice carrying a targeted disruption of BMP receptor 1A (BMPR1A) (Andl et al., 2004; Ming Kwan et al., 2004). SMAD proteins, which constitute a family of eight members (SMAD1–8), serve as intracellular mediators for TGFb signaling (Heldin et al., 1997; Massague, 1998; ten Dijke and Hill, 2004). SMAD2 and 3 respond to TGFb and activins, SMAD1, 5 and 8 function in BMP signaling pathway, while SMAD4 is a common mediator for both signaling pathways (Heldin et al., 1997; Massague, 1998; ten Dijke and Hill, 2004). Loss of function mutations of SMAD4 has also been detected in pancreas cancer, colon cancer, gastric polyposis and adenocarcinomas (Hahn et al., 1996; Friedl et al., 1999; Howe et al., 1998). In mouse, loss of Smad4 results in lethality at mouse embryonic E6–7 due to impaired extraembroynic membrane formation and decreased epiblast proliferation (Sirard et al., 1998; Yang et al., 1998). Using the Cre-loxP-mediated tissue specific knockout approach to overcome embryonic lethality, we showed that mice lacking Smad4 in their mammary glands displayed abnormal differentiation of mammary epithelium and tumorigenesis (Li et al., 2003b). Mean- while, Smad4 heterozygous mice developed gastric polyposis and cancer (Redman et al., 2005; Takaku Received 31 May 2005; revised 19 July 2005; accepted 19 July 2005; published online 19 September 2005 Correspondence: Dr X-J Wang, Department of Otolaryngology, Oregon Health and Science University, Portland, OR 97239, USA or Dr C-X Deng, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bldg. 10 Rm. 9N105, Bethesda, MD 20892, USA. E-mails: wangxiao@ohsu.edu or chuxiad@bdg10.niddk.nih.gov or chuxiad@mail.nih.gov Oncogene (2006) 25, 207–217 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc