Faculty Bibliography

The processing of tyrosinase, which catalyzes the limiting reaction in melanin synthesis, was investigated in melan-p1 melanocytes, which are null at the p locus. Endoglycosidase H digestion showed that a significant fraction of tyrosinase was retained in the endoplasmic reticulum. This retention could be rescued either by transfection of melan-p1 cells with an epitope-tagged wild-type p transcript or by treatment with either bafilomycin A1 or ammonium chloride. We found that the endoplasmic reticulum contains a significant amount of p protein, thus supporting a role for p within this compartment. Using immunofluoresence, we showed that most mature full-length tyrosinase in melan-p1 cells was located in the perinuclear area near the Golgi, in contrast to its punctate melanosomal pattern in wild-type melanocytes. Expression of p in melan-p1 cells restored tyrosinase to melanosomes. Triton X-114 phase separation revealed that an increased amount of tyrosinase was proteolyzed in melan-p1 cells compared with wild-type melanocytes. The proteolyzed tyrosinase was no longer membrane bound, but remained enzymatically active and a large proportion was secreted into the culture medium of melan-p1 cells. We conclude that p regulates posttranslational processing of tyrosinase, and hypopigmentation in melan-p1 cells is the result of altered tyrosinase processing and trafficking

BACKGROUND: The Ink4a-Arf tumor suppressor locus encodes two growth inhibitors, p16 and Arf, both of which are also implicated as effectors in cellular senescence. Because human germline defects in the INK4A-ARF locus are associated with familial melanoma, melanocytes may have unusual INK4A-ARF functions or controls of cell senescence. Because senescence is believed to be an anticancer mechanism, we investigated the role of Ink4a-Arf and its individual components in melanocyte senescence. METHODS: Melanocytes were cultured from littermate mice with zero, one, or two functional copies of the Ink4a-Arf locus. Senescence was evaluated by cumulative population doubling curves and by the assessment of acidic beta-galactosidase (an indicator of senescence) expression. Pigmentation and cell size were evaluated by spectrophotometry and microscopy. p16 and Arf expression in primary and spontaneously immortalized melanocyte or melanocyte precursor cell lines were evaluated by immunoblotting. Retroviral vectors containing normal p16 and Arf complementary DNAs were used to restore expression of these genes in Ink4a-Arf(-/-) melanocytes. RESULTS: Wild-type melanocytes (i.e., Ink4a-Arf(+/+)) senesced within 4-5 weeks of culture. Ink4a-Arf(-/-) melanocytes did not senesce and readily became immortal. Ink4a-Arf(+/-) melanocytes showed defective senescence. Senescent Ink4a-Arf(+/+) melanocytes were heavily pigmented, but Ink4a-Arf(+/-) and Ink4a-Arf(-/-) melanocytes were less pigmented. All of six spontaneously immortalized melanocyte or melanocyte precursor lines from Ink4a-Arf(+/+) mice lacked p16 protein expression, although most retained Arf protein expression. After restoration of p16 but not Arf expression, Ink4a-Arf(-/-) melanocytes stopped growing, became highly melanized, and expressed acidic beta-galactosidase. By contrast, restoration of Arf but not p16 expression led to cell death without evidence of senescence. CONCLUSION: Normal mouse melanocyte senescence and associated pigmentation require both copies of Ink4a-Arf and appear to depend more on p16 than on Arf function. Mutations of the INK4A-ARF locus may favor tumorigenesis from melanocytes by impairing senescence, cell differentiation, and (where ARF is disrupted) cell death

We report an instance of congenital granular cell tumors localized to the arm of a female infant. While granular cell tumors are well described during infancy as congenital epulis of the oral cavity, this case is unusual in both its location and histologic characteristics. The lesions, located around the antecubital fossa, were comprised of CD34-positive, S-100-negative granular cells. In addition, there were numerous eccrine glands in the upper dermis. The salient features of the case are discussed and reviewed in the context of the literature pertaining to this unusual entity

More than 10% of admissions worldwide to institutions for the visually impaired are due to some form of albinism. The most common form, oculocutaneous albinism type 2, results from mutations at the p locus. The function of the p gene is yet to be determined. It has been shown that melanocytes from p -null mice exhibit an abnormal melanosomal ultrastructure in addition to alterations in activity and localization of tyrosinase, a critical melanogenic enzyme. In light of these observations, we examined tyrosinase trafficking in p -null vs wildtype mouse melanocytes in order to explore p function. Electron microscopy of wildtype melan-a and p -null melan-p1 cells demonstrated accumulation of tyrosinase in 50 nm vesicles throughout the cell in the absence of p, an observation corroborated by an increase in tyrosinase activity in vesicle-enriched fractions from melan-p1 compared to melan-a cells. Misrouting in the absence of p was not limited to tyrosinase; a second melanosomal protein, tyrosinase-related protein 1, also trafficked incorrectly. In melan-p1, mislocalization led to secretion of tyrosinase into the medium. Adding tyrosine to the medium was found to partially correct tyrosinase trafficking and to reduce secretion; the cysteine protease inhibitor E64 also reduced secretion. We propose that p is required by melanocytes for transport of melanosomal proteins. In its absence, tyrosinase accumulates in vesicles and, in cultured melanocytes, is proteolysed and secreted.

To investigate the function of ocular albinism type 1 (OA1), the gene responsible for X-linked ocular albinism, we employed a construct containing murine Oa1 fused to green fluorescent protein (GFP) in a heterologous COS cell expression system. The cellular distribution of wild-type (WT) Oa1 protein and Oa1 proteins reflecting mutations causing X-linked ocular albinism were examined. Comparison with different organelle markers revealed that Oa1-GFP localized to the late endolysosomal compartments. Some Oa1 mutant proteins failed to exit the endoplasmic reticulum (ER) (Class I mutants), while other mutants partially (Class II mutants) or fully (Class III mutants) exited the ER and trafficked to endolysosomal compartments. We observed that expression of WT Oa1-GFP in COS cells caused an apparent enlargement of late endosomes and a redistribution of the mannose-6-phosphate receptor (M6PR). None of the mutants displayed the full range of effects on the redistribution of M6PR exhibited by WT Oa1. The effects of Oa1 on late endosome structure and content are thus likely to reflect an important biological property of Oa1. We propose that OA1 is involved in reorganizing the endolysosomal compartment as a necessary step in ocular melanosome biogenesis

To gain insight into the role of Oa1, the mouse homolog of the human X-linked ocular albinism 1 protein, its properties and subcellular localization were investigated. Antiserum raised against an expressed segment of the Oa1 protein recognized a band of approximately 48 kDa in immunoblots of extracts of cultured mouse melan-a melanocytes, but not of cells of non-melanocyte origin. When melanocyte extracts were treated with glycopeptidase F, a approximately 44 kDa band appeared. Like the melanogenic enzyme tyrosinase, expression of Oa1 was stimulated by alpha-melanocyte stimulating hormone and inhibited by agouti signal protein. Upon density gradient centrifugation of organelles of melan-a cells, Oa1 protein colocalized with the late endosomal/lysosomal marker Lamp1, but only partial overlap was observed with melanosomal proteins in the high density region of the gradient. Immunofluorescence staining revealed that neither endogenous Oa1 nor an Oa1-green fluorescent protein fusion product colocalized with the melanosomal protein tyrosinase related protein-1 in the cell periphery. In contrast, colocalization of Oa1 and Oa1-green fluorescent protein fusion product with Lamp1 was extensive throughout the cell. These results indicate that Oa1 is a melanocyte-specific integral membrane glycoprotein localized to late endosomes/lysosomes but not mature melanosomes. Considering the microscopic findings in patients with X-linked ocular albinism 1, we speculate that Oa1 may play a role in the trafficking of vesicles to developing melanosomes.