Several PATCHED1 Missense Mutations Display Activity in patched1-Deficient Fibroblasts

Abstract

Mutations in mouse and human patched1 (ptc1) genes are associated with birth defects and cancer. Ptc1 is a receptor for Hedgehog (Hh) signaling proteins. Hh proteins activate transcription of target genes, including ptc1, and Ptc1 represses those genes, both by regulating the activity of Gli transcription factors. We have established mammalian cell lines with reduced Ptc1 function and a lacZ reporter to investigate Hh signal transduction. Embryonic fibroblasts were derived from mice, heterozygous or homozygous for a ptc1 mutation that inserts lacZ under the control of the ptc1 promoter (ptc1-lacZ). In heterozygous ptc1 cells, ptc1-lacZ was expressed at low levels but could be induced by Sonic Hedgehog (Shh) and Gli-1. Homozygous ptc1 cells expressed high levels of ptc1-lacZ without Hh stimulation. ptc1-lacZ expression was dependent on cell density in ptc1 homozygotes and Hh-stimulated heterozygotes but was independent of density when Gli1 was used to activate ptc1-lacZ. A wild-type ptc1 transgene introduced into homozygous ptc1 cells greatly reduced ptc1-lacZ expression. Expression of either half of Ptc1 alone resulted in improper maturation of the protein and a failure to complement the ptc1 ؊/؊ cells. When co-expressed, both Ptc1 halves matured and had an activity similar to that of the intact protein. Three missense PTCH1 mutations exhibited significant functions in homozygous ptc1 cells. The missense mutants retained activity when expressed at about 10-fold lower levels and appeared as stable as wild-type Ptc1. These studies suggest that some tumors and disease phenotypes may arise from small reductions in PTCH1 activity.

Heterozygous PTCH1 Mutations Impact the Bone Metabolism in Patients With Nevoid Basal Cell Carcinoma Syndrome Likely by Regulating SPARC Expression

“…PTCH1 protein truncation mutations (observed in 86% of the NBCCS cases) often manifest as PTCH1 haploinsufficiency, as neither the N-nor the C-terminal half of PTCH1 is functional alone. (38,39) PTCH1 missense mutations have also been reported to result in variable levels of PTCH1 function, with some (Q802L and P1111L) associated with significant retention of activity, some (eg, L346R) causing only modest reduction in activity, and others resulting in substantial loss of function (R280C, G495V, and D499Y). (40,41) Consequently, the missense mutations in the remaining 4 cases (NB5, 9, 11, and 12) may have resulted in the partial retention of function, as a result of which these cases exhibited intermediate levels of PTCH1 activity compared with the wild-type and haploinsufficient lines.…”

Constitutive activation of the shh–ptc1 pathway by a patched1 mutation identified in BCC

“…Previously, two other studies have examined the molecular basis of several human ptc1 mutants identified from NBCCS/ BCC patients (Bailey et al, 2002(Bailey et al, , 2003. Results from these studies demonstrated that ptc1 mutants unable to mature have reduced protein stability and are unable to inhibit endogenous ptc1 promoter activity.…”

The EWS/FLI1 oncogenic transcription factor deregulates GLI1

“…The probes for GLI1 and PATCHED1 have been previously described (Bailey et al, 2002). The b-actin exposures for the blots in Figure 1d have been previously published (Zwerner and May, 2001).…”