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L M Milstone

Publications and source records attributed to L M Milstone.

At least 19 recordsLinked to original sources
PubMed1992

Parathyroid hormone-related protein. Evidence for secretion of a novel mid-region fragment by three different cell types.

The cDNA-predicted amino acid sequence of parathyroid hormone-related protein (PTHrP) contains multiple basic amino acid motifs, suggesting that PTHrP undergoes extensive post-translational processing prior to secretion. The secretory forms of the peptide are currently unknown. To identify these secretory forms, medium was harvested from three cell types: human renal carcinoma (SKRC-1) cells, human keratinocytes, and rat insulinoma cells stably transfected with the cDNA for PTHrP(1-141) (RIN-141 cells). Amino-terminal species were immunopurified using an anti-PTHrP(1-36) column, and mid-region species using an anti-PTHrP(37-74) column. PTHrP peptides in medium and in cell extracts were further resolved by reverse phase high performance liquid chromatography (RP-HPLC) and identified using region-specific immunoassays. SKRC-1 and RIN-141 cells secreted three distinct amino-terminal species and a novel, non-amino-terminal, mid-region fragment. Sequence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the RIN-141 cell mid-region fragment begins at amino acid 38 of the cDNA-predicted sequence and is approximately 70 amino acids in length. Comparison of RP-HPLC elution patterns suggests that SKRC-1 cells and keratinocytes secrete a similar or identical mid-region fragment. Immunofluorescence studies revealed a Golgi pattern for the amino-terminal species and a secretory granule pattern for the mid-region fragment. These studies indicate that 1) multiple PTHrP species are secreted, including a novel mid-region fragment; 2) Arg37 serves as a cleavage site in at least three cell types; 3) PTHrP(1-36) is likely to be an authentic secretory form of PTHrP; and 4) the mid-region fragment appears to be packaged into secretory granules. The marked interspecies conservation of this mid-region PTHrP suggests that it will have important biological functions.

Amino Acid Sequence↗
PubMed1992

Methotrexate induces differentiation of human keratinocytes.

Terminal differentiation is a key element in the maintenance of tissue homeostasis in the epidermis. We show here that methotrexate (MTX) induces differentiation of human epidermal keratinocytes in vitro. MTX inhibits proliferation of keratinocytes and also induces several markers of differentiation: a change in cell morphology, a marked increase in cell size, an increase in the proportion of cells that express involucrin, and an increase in the amount of cornified envelope protein. These effects of MTX are dose- and exposure-time-dependent and become irreversible after 24 hr, approximately one population doubling time. These effects of MTX cannot be attributed to cytotoxicity since keratinocytes not only remain viable but also actively synthesize proteins. MTX causes reproducible changes in the SDS/PAGE profiles of newly synthesized proteins and, in particular, increases the amount of involucrin synthesis. Thymidine completely prevents these effects of MTX, suggesting that they are caused by a depletion of thymine deoxyribonucleotides. The effect of MTX on keratinocytes may provide a model for studying the relationship between deoxyribonucleotide metabolism and differentiation in normal cells. In addition, the ability of MTX to induce differentiation in keratinocytes suggests a mechanism to explain its therapeutic action in psoriasis.

Cell Differentiation↗
PubMed1992

The core protein of epican, a heparan sulfate proteoglycan on keratinocytes, is an alternative form of CD44.

Epican, a heparan sulfate proteoglycan, was recently identified on the surface of keratinocytes with the aid of a monoclonal antibody to its core protein. Using that antibody to screen a human keratinocyte cDNA library, a clone encoding the entire epican core protein was selected and sequenced. The core protein of epican is a form of CD44. The deduced protein sequence of 699 amino acids has a novel 339 amino acid domain inserted into the proximal extracellular domain of the standard, leukocyte form of CD44. The additional domain adds a number of potential N- and O-linked glycosylation sites and two proteolysis sites to this form of CD44.

Amino Acid Sequence↗
PubMed1992

Identification and characterization of a cell surface proteoglycan on keratinocytes.

Proteoglycans fill the intercellular space between keratinocytes but their structure and function are not well understood. We have identified and partially characterized one intercellular proteoglycan on human keratinocytes, for which we propose the name epican (epidermal intercellular proteoglycan). Monoclonal antibodies (MoAb) were generated from a mixture of keratinocyte proteoglycans. One, designated MoAb17, identified the core protein of an intercellular proteoglycan that had an apparent mobility of greater than 250 kDa on Western blots. The core protein itself had an apparent mobility of 180 kDa following deglycosylation with trifluoromethanesulfonic acid. Enzymatic deglycosylation revealed that most core protein molecules were substituted with heparan sulfate but that some carried chondroitin sulfate instead. Smaller forms of the core protein were more abundant in tissue-culture medium than in cell extracts. This proteoglycan was localized by immunofluorescence to the intercellular space of the epidermis and the surface of keratinocytes in vitro, particularly at cell-cell contacts. MoAb17 did not react with protoglycans extracted from other skin cells, nor did it bind to basement membranes or connective tissue. Comparison of Western immunoblots using MoAb17 and antibodies to core proteins of other proteoglycans suggested that epican is not related to syndecan but is a member of the CD44 family.

Antibodies, Monoclonal↗
PubMed1992

Analysis of PTHRP binding and signal transduction mechanisms in benign and malignant squamous cells.

We have explored a potential autocrine role for parathyroid hormone-related protein (PTHRP) in malignant squamous carcinoma cells (SqCC) and their nonmalignant counterpart, human epidermal keratinocytes (HK). Specific binding of Tyr36 human PTHRP-(1-36)NH2 (125I-[Tyr36]hPTHRP-(1-36)NH2) was identified in 75% of unselected SqCC lines. In contrast, no binding was detected on the mouse keratinocyte line BALB-MK or on five different HK lines. Although each SqCC and keratinocyte line secreted immunoreactive PTHRP into its medium, there was no correlation between PTHRP concentration and number of binding sites. Inhibition of binding by [Tyr36]hPTHRP-(1-36)NH2 yielded half-maximal inhibitory concentration values of approximately 100 nM in all SqCC lines. Affinity cross-linking of SqCC cells revealed 98- and 70-kDa binding proteins with similar affinity (approximately 100 nM). Exposure of fura-2-loaded SqCC cells to PTHRP and PTH resulted in equivalent, dose-dependent transient increases in intracellular calcium [half-maximal effective concentration (EC50) = 0.08 nM]. PTHRP also increased intracellular calcium in HK (EC50 = 0.05 nM). No adenosine 3',5'-cyclic monophosphate (cAMP) response to PTHRP or PTH was elicited in either SqCC or HK, despite brisk isoproterenol responses in both. We conclude that high-capacity low-affinity binding sites for PTHRP are detectable in the majority of SqCC lines but not in HK. These low-affinity binding sites are unlikely to represent receptors. The sensitive intracellular calcium response suggests the additional presence of high-affinity receptors on SqCC as well as on HK. However, the failure of PTHRP or PTH to stimulate cAMP production in otherwise cyclase-competent cells suggests that these are not classical PTH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1992

Serum parathyroid hormone level is elevated in some patients with disorders of keratinization.

BACKGROUND AND DESIGN: After the chance of observation of an elevated parathyroid hormone (PTH) value in a patient with pityriasis rubra pilaris, the serum PTH level was measured in the next 14 patients seen with disorders of keratinization. Calcium metabolism in three affected patients was then studied in depth. RESULTS: Five of 15 patients had twofold or greater elevations in serum PTH values. The patients had four different disorders of keratinization: bullous congenital ichthyosiform erythroderma (two patients); lamellar ichthyosis (one patient); pityriasis rubra pilaris (one patient); and ichthyosis linearis circumflexa (one patient). At least one other patient with each diagnosis had normal PTH values. Two of three patients who were studied further had clear evidence of increased, biologically active PTH, consistent with secondary hyperparathyroidism. An elevated PTH level spontaneously became normal in one patient, and in a second patient it became normal with a high-calcium diet. CONCLUSIONS: These data provide the first indication that patients with various disorders of keratinization have an increased risk for secondary hyperparathyroidism. The exact prevalence, origin, and physiologic significance of this finding remain to be elucidated.

Adolescent↗
PubMed1992

Human dermatosparaxis: a form of Ehlers-Danlos syndrome that results from failure to remove the amino-terminal propeptide of type I procollagen.

Dermatosparaxis is a recessively inherited connective-tissue disorder that results from lack of the activity of type I procollagen N-proteinase, the enzyme that removes the amino-terminal propeptides from type I procollagen. Initially identified in cattle more than 20 years ago, the disorder was subsequently characterized in sheep, cats, and dogs. Affected animals have fragile skin, lax joints, and often die prematurely because of sepsis following avulsion of portions of skin. We recently identified two children with soft, lax, and fragile skin, which, when examined by transmission electron microscopy, contained the twisted, ribbon-like collagen fibrils characteristic of dermatosparaxis. Skin extracts from one child contained collagen precursors with amino-terminal extensions. Cultured fibroblasts from both children failed to cleave the amino-terminal propeptides from the pro alpha 1(I) and pro alpha 2(I) chains in type I procollagen molecules. Extracts of normal cells cleaved to collagen, the type I procollagen synthesized by cells from both children, demonstrating that the enzyme, not the substrate, was defective. These findings distinguish dermatosparaxis from Ehlers-Danlos syndrome type VII, which results from substrate mutations that prevent proteolytic processing of type I procollagen molecules.

Amino Acids↗
PubMed1991

Quantitation of cross-linked protein: an alternative to counting cornified envelopes as an index of keratinocyte differentiation.

Covalently cross-linked protein is the key feature of the cornified envelope, an important marker of squamous differentiation. Enumeration of cornified envelopes is widely used to assess keratinocyte differentiation, but is tedious and subjective. We report here a rapid, objective, sensitive, and quantitative assay that measures total cross-linked protein in keratinocytes. The method is based on the resistance of cross-linked protein to solubilization in boiling SDS-beta ME, separation of cross-linked from soluble protein by collection of cross-linked protein on sheets of regenerated cellulose, binding of Coomassie blue to protein, and quantitation by scanning laser densitometry. There is excellent correlation between the cross-linked protein measured by this assay and the number of cornified envelopes.

Cell Differentiation↗
PubMed1991

Immunoaffinity purification of parathyroid hormone-related protein from bovine milk and human keratinocyte-conditioned medium.

Parathyroid hormone-related proteins (PRHrP) are a novel family of proteins that appear to be responsible for humoral hypercalcemia of malignancy. Although PTHrP derived from human tumors have been purified and their N-terminal amino acid sequence determined, and although the structure of the PTHrP gene and its alternatively spliced mRNA transcripts have been defined, the secretory and circulating form(s) of the protein are unknown. Purification of PTHrP in the past has been difficult, requiring multiple chromatographic steps and months or years to complete. To define naturally occurring PTHrP species we have developed a rapid and efficient immunoaffinity purification method. Bovine milk (250 ml) and human keratinocyte-conditioned medium (3000 ml) were affinity purified using a 300 microliters affinity-purified polyclonal anti-PTHrP-(1-36) antibody column and a single RP-HPLC step. Purification required only 7-10 days and yielded a 3-4% recovery. Quantities of PTHrP sufficient for silver-stained SDS-PAGE, Western analysis, and N-terminal amino acid sequence were obtained. In contrast to conventional purification schemes, affinity purification of PTHrP is rapid and efficient and can be applied to biologic samples that contain PTHrP in low abundance. These methods can be applied to the purification and characterization of the as yet undefined secretory and circulating forms of PTHrP.

Animals↗
PubMed1991

Immunohistochemical localization of parathyroid hormone-related protein (PTHRP) in normal human skin.

Human keratinocytes secrete large amounts of a parathyroid hormone-related peptide (PTHRP) in vitro. Because recent studies indicate that PTHRP could have a number of autocrine or paracrine functions in the skin, localization of this peptide in vivo is important. A monoclonal and two affinity-purified polyclonal antibodies were employed to locate PTHRP in normal human skin and cultivated human keratinocytes. PTHRP is present throughout the viable portion of the epidermis, in adnexal epithelial cells, and in all cultivated keratinocytes. These findings do not support the provocative suggestion that PTHRP is a marker for squamous differentiation.

Antibodies↗
PubMed1991

Antiviral activity of 5-iodo-2'-deoxyuridine and related drugs in human keratinocytes infected in vitro with herpes simplex virus type 1.

5-Iodo-2'-deoxyuridine (IUDR) is a potent topical antiviral agent in experimental animals but is less active in man for treating cutaneous viral infections. We have shown here that IUDR is 5 times less active in human keratinocytes infected in vitro with herpes simplex virus type 1 than in guinea pig embryo cells infected in culture. To account, in part, for this difference in activity of IUDR, we measured the capacity of these different cultures to catabolize and thus inactivate the drug. IUDR is catabolized by thymidine phosphorylase; activity of this enzyme was very high in human keratinocytes in vitro but was very low in guinea pig embryo cells. The antiviral activity of IUDR in human keratinocytes, however, was not increased by inhibiting thymidine phosphorylase; inhibiting thymidine phosphorylase apparently increased the availability of thymidine that would compete with IUDR and, indeed, the activity of IUDR in infected cells was reduced by addition of thymidine to the medium. These data indicate that the catabolism of IUDR and related analogs alters antiviral activity in human keratinocytes.

Animals↗
PubMed1991

Glycosylation of parathyroid hormone-related peptide secreted by human epidermal keratinocytes.

While the gene and mRNA transcripts encoding PTH-related peptide (PTHrP) have been well characterized, the actual secretory form(s) of the peptide is unknown. Accordingly, synthetic and recombinant PTHrPs employed to date for biological and immunological characterization have necessarily been of arbitrary lengths. No prior evidence for glycosylation of PTHrPs has been described. To define the naturally occurring form(s) of this peptide secreted by human epidermal keratinocytes, we have affinity purified, using an anti-PTHrP-(1-36) antibody column, human PTHrP secreted under conditions of protease protection. Human keratinocyte-conditioned medium collected without measures to protect against proteolytic degradation contains multiple PTHrP immunoreactive and bioactive species. In contrast, under conditions of protease protection, human keratinocyte-conditioned medium contains a single 18,000 mol wt (Mr) form of the peptide. In contrast to recombinant and synthetic PTHrPs, which migrate as distinct, well focussed bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this 18,000 Mr PTHrP displays the broad electrophoretic profile of a glycoprotein. Treatment of this peptide with trifluoromethanesulfonic acid, an agent that deglycosylates both O- and N-linked saccharides from their core proteins, shifted the Mr of the protein to approximately 10,000. In contrast, exposure of recombinant PTHrP-(1-141) to the same agent results in no change in electrophoretic mobility. These studies indicate that the 18,000 Mr species of PTHrP secreted by human epidermal keratinocytes is a glycoprotein.

Blotting, Western↗
PubMed1989

Dipyridamole potentiates the growth-inhibitory action of methotrexate and 5-fluorouracil in human keratinocytes in vitro.

Human keratinocytes transport extracellular thymidine across the plasma membrane and incorporate it into DNA. Data presented here show that dipyridamole, a well-known inhibitor of facilitated diffusion of nucleosides, blocks the transport of thymidine into human keratinocytes in vitro. Dipyridamole (1.0 microM) inhibited the transport of 3H-thymidine (0.2 microM) into intracellular material by 75% and its subsequent salvage and incorporation into DNA by 48%. Dipyridamole (1 microM) did not affect the growth of keratinocytes in vitro but did potentiate the growth inhibition caused by methotrexate (MTX) or 5-fluorouracil (5-FU). The growth of keratinocytes exposed to 0.1 microM MTX for 8 d was inhibited by 32%. However, in combination with a noninhibitory concentration of dipyridamole (1 microM), this concentration of MTX (0.01 microM) inhibited the growth of keratinocytes by 93%. Thymidine in culture medium reversed the cytotoxicity of MTX. However, in the presence of dipyridamole, thymidine in the culture medium did not reverse the action of MTX. The synergistic interaction between MTX and dipyridamole was also observed with 5-FU and dipyridamole. 5-FU (0.5 microM) inhibited cell growth by 30% but in combination with dipyridamole (1 microM), inhibited cell growth by 86%. These data are consistent with the theory that inhibiting thymidine salvage by blocking transport of extracellular thymidine potentiates the growth inhibitory action of inhibitors of de novo pyrimidine biosynthesis in human keratinocytes. Combination chemotherapy, such as methotrexate plus dipyridamole, might be efficacious in the treatment of hyperproliferative diseases of the epidermis.

Cell Division↗
PubMed1989

Native and a synthetic analogue of the malignancy-associated parathyroid hormone-like protein have in vitro transforming growth factor-like properties.

A human parathyroid-like protein (PLP) has recently been isolated and cloned from human tumors associated with the paraneoplastic syndrome, humoral hypercalcemia of malignancy. PLP shares NH2-terminal amino acid sequence similarity with PTH but has a unique primary structure thereafter. Studies reported to date have indicated that both native and synthetic amino-terminal PLP polypeptides display actions in vivo and in vitro that are similar to those of PTH. We report here that purified native PLP and synthetic 36Tyr(1-36)amide human PLP induce epidermal growth factor-dependent transformation of NRK 49F cells in soft agar. Further, the synthetic peptide induces a significant increase in the biosynthesis of fibronectin by human dermal fibroblasts. (1-34)PTH does not display either of these biological activities. These data indicate that there are qualitative differences between PTH and the recently identified PLP. The latter hormone appears to possess transforming growth factor-like properties that may be relevant to its physiological actions.

Cell Transformation, Neoplastic↗
PubMed1988

Human keratinocytes catabolize thymidine.

Human neonatal foreskin keratinocytes incorporate exogenous thymidine into DNA and proliferate in vitro even after reaching confluence. Keratinocytes also catabolize thymidine, as reported for the first time below. Stratified cultures of keratinocytes reduced the amount of thymidine in the medium by more than 90% within 2 to 4 h. Consequently, the rate of incorporation of thymidine (0.2 microM, 4 microCi/ml) into DNA was linear for no more than 2 h. Linear incorporation of thymidine into DNA for at least 12 h could be achieved by continual addition of fresh radioactive thymidine to the culture medium. Different tissues have widely differing abilities to catabolize thymidine. Cutaneous catabolism of thymidine shows striking species differences. Soluble extracts from human neonatal foreskin and adult skin, as well as from cultivated human keratinocytes, actively catabolize thymidine. Soluble extracts of skin from mouse, rabbit, or guinea pig do not catabolize thymidine. Extracts from cultivated human fibroblasts and melanocytes have little or no ability to catabolize thymidine. Catabolism of thymidine by keratinocytes has important implications for the use of [3H]thymidine in studies of keratinocyte proliferation and for the use of thymidine analogs in therapy of cutaneous disease.

Cells, Cultured↗